Not much is known about Tamayo's lifestyle when she was a human except that she was a married woman with children.
Shortly before she became a Demon, Tamayo fell ill with a life-threatening disease and began seeking a cure, which is how she first came across Muzan Kibutsuji. Hiding his true intentions, Muzan offered to treat Tamayo's illness and, when she naively accepted, swiftly converted her into a Demon and left her to run rampant around the town. It's noted that she killed not only her husband and children while on this rampage, but also a number of other humans after the realization of what she'd done to her family sunk in and she fell into a void of despair.
Although the exact order of events are unknown,eventually it was seen that Tamayo had seemingly become a subordinate to Muzan and had seemingly become subservient to the demon progenitor, as seen during Muzan's confrontation and subsequent battle against Yoriichi Tsugikuni. Where Tamayo is seen quietly standing beside Muzan in complete servitude as he bantered with the Demon Slayer.
This likely hints that at one point, Tamayo had attempted to kill Muzan and avenge her family and him turning her into a Demon, a battle she likely lost and her having her spirit completely broken by the Demon Lord due to seeing the massive difference in power between herself and him,eventually leading to her becoming his servant in defeat.
However this all changed upon seeing the battle between Muzan and Yoriichi,where upon seeing the Demon Slayer completely overpowering and nearly dealing a death blow on the Demon Progenitor, Tamayo was seen to have been completely dazzled by the sheer power of the Demon Slayer and upon seeing Muzan defeated and near death, her spirit to kill and defeat him was seemingly reignited.
After Muzan's escape by scattering himself to pieces, Tamayo was shown to have reacted in visible anger at the Demon Progenitor's escape and expressed frustration at him seemingly obtaining immunity to decapitation. These emotions, along with the glint of hope he saw in her eyes as she saw Muzan's near death and defeat,prompted Yoriichi to spare Tamayo and console her due to realizing their mutual hatred for Muzan. Eventually leading to him allowing her to leave with her life,despite knowing of the repercussion from the rest of the Demon Slater Corps for not only failing to kill Muzan, the demon progenitor and the main enemy of the Demon Slayers, but for also allowing a Demon as powerful as Tamayo to escape.
After a period of time, Tamayo regained her sanity and proceeded to set up a life in which she could study treatments for Demonic cures. After about 200 years of study, she came across a sick young boy with nowhere to go, and successfully converted him into a Demon with his consent. This boy became known as Yushiro, and he has remained her faithful and protective assistant ever since.
As a trio of policemen attempt to pull Tanjiro Kamado off of a recently transformed Demon, Tamayo uses Blood Bewitchment: Aroma of Visual Fantasy to confuse their senses, then approaching the Demon Slayer alongside her partner, Yushiro. She comments on the fact that Tanjiro was still addressing the Demon in a human way, and trying to save him, and decides to lend him a hand. Confused by the contrast of her Demonic smell to her human personality, the Demon Slayer begins to question Tamayo, and she explains that, even though she'd a Demon, she's also a doctor, and she shares the same hatred for Muzan as Tanjiro does and also wishes to eliminate him.
Later, at her hideout, Tamayo welcomes Tanjiro to her abode, explaining the status of the earlier Demon and his wife to the Demon Slayer and reprimanding Yushiro for resorting to violence at Tanjiro's questions. She then introduces herself and her colleague, answering Tanjiro's earlier question by explaining her unique ability to manipulate her body at whim, as well as her and Yushiro's shared needing of only a little human blood to survive. She further explaines that she is the one who transformed Yushiro into a Demon, and reprimands the latter once more for getting violent with Tanjiro. Tamayo informs Tanjiro that she wasn't trying to increase the Demon population, instead offering a transformation as a choice to those who were dying.
Tanjiro then asks her if there's a way to turn Demons back into humans, to which she answers that there may well be one. After reprimanding Yushiro once again for his overly violent behavior, she explains that she currently cannot hope to perform this procedure, as first she needs to research and develop it. Tamayo says that Tanjiro must allow her two things in order to create this remedy: the blood of strong Demons, such as the Twelve Demon Moons, who have a large concentration of Muzan's own blood flowing through their own, and a chance to study his sister's blood. She exlplains and reiterates the danger of her first request to Tanjiro as a warning, and then asks him if he's still up for the job, to which he smiles and accepts her request, saying that this quest would be a great step forward for recovering the lives of many other people, not just Nezuko.
Suddenly, a large handball tears through the room, and Yushiro jumps in front of Tamayo in an attempt to protect her from it. Susamaru appears, revealed to be the owner of the handball, and begins attacking the four with more balls, throwing them to hard that they ricochet off the walls, one even slicing Yushiro's head clean off. Tamayo clutches the body of her partner as he slowly regrows his head, and tells Tanjiro and Nezuko not to worry about the two of them and to carry on with their own battle, since, as they're Demons, they can fend for themselves. Tanjiro accepts this and begins to fight back against Susamaru, while Yushiro, now that his head has grown back, begins to curse the Demon Slayers who take away his time with Tamayo as they only leave more traces and make it easier for Muzan to find the pair of them, since Yushiro's cloaking technique is still imperfect.
The two move as far from the center of the battle as possible, and Tamayo yells once again to Tanjiro that he mustn't worry for them, while Yushiro gives him advice for what to do against the Demon's handballs and lends him his Demon sight.
Shortly after, as the battle draws somewhat to a close, Tamayo approaches Susamaru, asking her if she knew Muzan's true identity and telling her that he's nothing but a coward. Susamaru attempts to silence her, but Tamayo ignores her and goes on to state that Demons can't live together and have to kill each other purely because Muzan is manipulating them, trying to keep them from grouping together so they can't stage an attack on him. As Susamaru desperately attempts to silence her, Yushiro notes that Tamayo is using her Blood Bewitchment: Mystical Fragrance of the White Day ability on the Demon to get her to say Muzan's name, which she does, and Muzan's curse then suddenly begins to break out from inside her.
Susamaru is swiftly killed by the curse as Tanjiro and Yushiro watch in horror, and Tamayo goes over to her body to inspect her eyes. Since no words have been carved into them, Tamayo recognizes that Susamaru was not, in fact, one of the Twelve Demon Moons, and explains this and the curse to a bewildered and shocked Tanjiro. Emotionlessly, Tamayo collects blood samples from the Demons and heads back inside her house.
After Tanjiro has recovered a little from his battle, he is invited to Tamayo's basement, where Nezuko, having been sheltering there from the new morning sun, runs over to him and hugs him, then runs back to Tamayo and throws her arms around her as well, also reaching out a hand to pat an embarassed Yushiro on the head. Somewhat confused, Tamayo explains that Nezuko has been like this towards them ever since the battle ended, and Tanjiro smiles and says that it must be because she recognizes them as part of her family since, due to their humane personalities, Nezuko sees them as people she must protect, even though they're actually Demons. Touched by this, and still in Nezuko's embrace, Tamayo begins to tear up and buries her face into the young Demon's shoulder, thanking her.
A short while later, as Tanjiro and Nezuko prepare to say their goodbyes to the good Demons, Tamayo explains that she and Yushiro will be leaving their house as it's too close to Muzan, and she also suspects she will be exposed as a Demon rather soon. She then asks the Demon Slayer if he wishes to leave his younger sister with them, since her being in Tamayo's care will almost guarantee her survival and protection, but, when a determined Nezuko grabs his hand, Tanjiro denies her offer. Tamayo smiles, understanding his decision, and wishes him the best on his travels as he and his sister leave their house.
Using blood samples from Demon Moon members Kyogai, Daki, and Gyutaro Tanjiro provided for her, Tamayo was able to synthesize an antidote for Demonism, which she successfully tested on one of Muzan's victims. She later wrote about her progress to Tanjiro in a letter. In it, she also expressed her amazement about Nezuko's blood samples he had given her, which had gone through multiple mutations. This led her to speculate that the reason why she had not regained her intelligence yet is because she is prioritizing developing a resistance to the sun; a theory proven true when Nezuko becomes the first Demon in history immune to sunlight.
Tamayo appears once again after Muzan's attack on the Ubuyashiki residence, shortly after Kagaya Ubuyashiki, alongside his wife and two of his daughters, blew Muzan and themselves up. As Muzan tries to recover from the wounds he received during the explosion, Tamayo releases her Flesh Seed attack, sending large, thorn-like spikes out to pierce through his body. Muzan realizes that he's been immobilized by a Blood Demon Art and begins to try and absorb the spikes, but Tamayo suddenly rushes towards him and pushes her fist through his stomach. Bewildered, Muzan asks her why she's here and considers what her goal might be in doing this, and she explains that her fist contains a drug that can turn a Demon back into a human. An expression of shock forms on Muzan's face as he voices his denial of that possibility, but Tamayo just smiles menacingly and says that it's definitely real, although she didn't achieve it alone.
Muzan says that Tamayo is one stubborn woman and that her hatred towards him is unjust, since it wasn't him who killed her family, it was herself. Now crying, Tamayo screams that she wouldn't have become a Demon if she knew that would be the outcome, since all she wanted was to recover from her disease so she could see her kids grow up. As Muzan places his hand on Tamayo's head and begins to crush her eye with his thumb, to reopen the wounds further, Muzan goes on to remind the Demon of how she killed even more humans after her family as she fell into despair, which Tamayo then admits to. She says that that is why she must pay for her sins, and why both her and Muzan were going to die right there, then yelling for Gyomei Himejima to attack her and Muzan with the intention to kill. The Stone Hashira swings his spiked flail at the Demons, knocking Muzan's head clean off. However, the Demon lord simply regenerates his lost limb, refusing to die, and Gyomei decides to resort to keeping him restrained until sunrise so he can let the sun disintegrate him.
Just then, while still clutching Tamayo's head and much to Gyomei's surprise, Muzan unleashes a black Blood Demon Art: Chain of Thorns. Gyomei quickly protects himself with his third breath style, and, right on cue, Sanemi Shinazugawa, Giyu Tomioka, Muichiro Tokito, Tanjiro Kamado, Shinobu Kocho, Mitsuri Kanroji and Obanai Iguro appear to join the battle.
Once Akaza, Doma, and Kokushibo have successfully been eliminated, it is revealed that Muzan has revived himself and evolved his body into a new form. After swiftly dealing with the Demon Slayer Squads sent after him, Muzan holds up the detached head of Tamayo. As he stares her down, he declares that the drug was a failure. Tamayo curses Muzan to hell, only for him to declare he will not be defeated, as hundreds of humans have said the same as her. In Tamayo's last moments, she pleas for Muzan to give her family back. He tells her to return to the side of the family she killed, crushing her skull afterwards. This is felt by Yushiro, who is seemingly sent into a rage by her death.