More than three hundred years ago, an alchemist named Makropulos was employed by the Hapsburg Emperor Rudolf II to create an elixir to give the Emperor eternal life. Not trusting Makropulos’s finished potion, the Emperor forced him to administer it to his own daughter, Elina. When she became seriously ill Makropulos was put in prison, but Elina eventually recovered and escaped. What was not apparent at the time was that the elixir actually worked, granting Elina some 300 additional years of life. During the time that she has lived, Elina has had many identities, many names (always with the initials E.M.), and many affairs. One of her more passionate affairs was with Baron Josef Ferdinand Prus, by whom she had a son. While Prus left his estate in writing to this illegitimate son, the will has long gone missing and there has ensued a century-long dispute between two branches of the family, Gregor and Prus, over rights to the estate.
At Dr. Kolenatý’s law office in Prague, the clerk Vítek is filing papers concerning the long-standing case of Gregor vs. Prus. Gregor, the plaintiff, enters to inquire about the case’s progress, followed shortly by Vítek’s daughter Kristina. She is a student of singing and speaks enthusiastically about the great prima donna, Emilia Marty.
Kolenatý enters his office with none other than Emilia Marty. She has come to learn about the Gregor vs. Prus case and displays a remarkable knowledge of it. Marty claims that Baron “Pepi” Prus, who was believed to have died childless in 1827, was in fact the father of Ferdinand Gregor, whose mother was the famous singer Ellian MacGregor. (As this could not be proven at the time of the death of Pepi Prus, the Prus estate passed to a cousin.) Marty claims to know of documents in the vaults of the current Baron Prus that will settle the matter conclusively. Kolenatý does not believe her and cannot imagine how she could know this information, but Gregor insists that he look into the matter.
After Kolenatý has left, Gregor offers Marty a reward if this information helps him to win his case. She scorns his offer of money but asks if he might procure for her a sealed envelope that she knows to be among the papers in Prus’s vault. Kolenatý returns along with Baron Prus, having found Pepi’s will, his love letters to Ellian MacGregor, and the sealed envelope. Prus points out that it is still necessary to produce written proof that Ferdinand MacGregor was indeed the son of Pepi Prus before Gregor can legally claim the disputed property. Marty announces that she can produce this, and leaves the men astonished once again.
After a performance, a stagehand and a cleaning woman discuss Marty’s singing. Prus comes backstage looking for Marty, and Kristina and Janek (Kristina’s sweetheart and the son of Prus) enter, as well as Gregor and Vitek who come to congratulate the singer. Marty’s directness and apparent lack of tact insults Janek and Gregor, yet when the half-senile Hauk-Šendorf enters and remarks on how much she reminds him of his love of fifty years past, the Spanish dancer Eugenia Montez; Marty speaks to him in Spanish and accepts him with open arms. Exhausted, she dismisses everyone but Prus, who proceeds to question her about other documents and love letters he has found in his vaults. He notes that since the love letters to Pepi Prus are only signed with the initials “E.M.,” it cannot be proven that Ellian MacGregor was indeed Ferdinand Gregor’s mother. Furthermore, he reveals that his mother gave her name as Elina Makropulos at Ferdinand’s baptism; Marty is astonished that Prus is on the trail of her true identity. She then realizes that the signed proof she sent Kolenaty that Ferdinand Gregor was the son of Ellian MacGregor is not valid. Desperate for the sealed envelope in Prus’s possession, Marty asks him to name his price for it, but he does not respond to her offer and leaves.
Gregor makes protestations of love to Marty, but in exhaustion she falls asleep. Gregor leaves and Marty awakens to find Janek staring at her in fascination. She asks him to steal the sealed envelope from his father, and he agrees. Prus catches them, dismisses Janek, and agrees to give her the document in exchange for a night of love.
It is early morning and Marty and Prus are in her hotel room. Marty demands the sealed envelope from him, and he angrily gives it to her. A maid enters and explains that Prus’s servant is downstairs with a message. He hurries out and returns with the news that his son, Janek, has just killed himself out of love for Marty. She coldly remarks that lots of people kill themselves and Prus leaves in a rage. Hauk-Šendorf enters ready to take Marty to Spain with him. Marty agrees and begins to pack, but they are interrupted by the arrival of Kolenatý, Gregor, Kristina, Prus, and Vitek. Kolenaty, suspicious that Marty is a fraud, produces the letter she sent him claiming to be from Ellian MacGregor, noting that it is in the same handwriting as that of Marty herself. Marty excuses herself, and while she is away, they search her personal belongings and discover correspondence bearing various names: Emilia Marty, Ellian MacGregor, Eugenia Montez, Elsa Mueller, Ekaterina Myshkin, and Elina Makropulos.
Marty returns with a bottle of whisky, which gives her courage to confess her real identity: she is actually Elina Makropulos, born in Crete in 1575. To the disbelief of everyone in the room, she explains how her father was made to administer the longevity potion to her as a child. She has lived ever since, in different countries under different names, but now feels her strength failing and requires the formula (the sealed envelope) to rejuvenate herself. She collapses and upon reviving realizes that she does not want the potion after all, for her life has been marked by boredom and loneliness. She offers the formula to Kristina and sinks happily into death.