A tourney has been given in honor of the French ambassador, who is negotiating with Elisabetta concerning a marriage proposal from the King of France, which she is contemplating through a sense of duty to her subjects, while secretly pining for Leicester, whose absence from court she notices. Talbot, who is in charge of the royal prisoner, tries to intercede with Elisabetta on behalf of Maria, imprisoned in Fotheringay Castle since fleeing from Scotland. But the Queen is torn between sympathy for Maria and fear that she is plotting against her; Cecil warns against the perils of pity.
Leicester arrives and she gives him a ring to take to the French Ambassador as a token of her acceptance of the offer of marriage, but is incensed when Leicester seems unmoved by the commission. Privately Talbot gives Leicester a portrait of Maria and a letter from her and Leicester resolves to free the woman he loves by any means. He gives Elisabetta the letter, which is a plea for a meeting with her, and he urges her to consent, pointing out that she can use a hunting party in the vicinity of Fotheringay as a pretext. His enthusiasm for her rival's cause reminds Elisabetta of Maria's attempts on the English throne and when Leicester waxes unwisely lyrical about Maria's charms, the Queen exults that she has been brought low.
Accompanied by Anna, Maria walks in the park, rejoicing in her limited freedom, but remembering sadly the happy days of her youth in France. The sound of the approaching royal hunt terrifies her, and she regrets having asked Elisabetta for a meeting, but, supported by Leicester and his assurances that Elisabetta had been moved by the letter, she agrees to stay and face her.
Elisabetta also views the occasion with mixed feelings, on the one hand rejecting Cecil's urgings that she execute Maria and on the other enraged by the fervor with which Leicester argues her rival's case. As the queens confront one another, each is already convinced that the other is haughty, but Maria makes an effort and humbles herself to ask for clemency. Elisabetta is obdurate, and her references to Maria's murdered husband and aspersions on her honor provoke Maria, despite Leicester's attempts to calm her, into taunting Elisabetta with being a bastard and a "vile, lascivious harlot." Furious, Elisabetta advises her to expect her death sentence, but Maria exults in her temporary triumph.
Although mortally affronted, Elisabetta hesitates to sign the death warrant, despite the urgings of Cecil that her safety and that of the realm depend on Maria's death. Only the arrival of Leicester provokes her into signing. His prayers for mercy only provoke her into ordering him to witness the execution.
Maria is still exultant over her humiliation of Elisabetta, though fearing that Leicester may be in danger from her wrath. Cecil brings the death warrant. She refuses his offer of a priest, but admits to Talbot that she is oppressed by the recollection of her sins. He reveals that he has taken holy orders so as to hear her confession.
She confesses to guilt over the murder of her husband, Darnley, and also seems to admit complicity in the Babington plot (not only to free her but elevate her to the English throne by murdering the Queen). Talbot gives her absolution.
Maria's friends lament her fate, and she, facing death calmly, tries to comfort them and give them strength. As the cannon sounds the signal for her execution, Cecil asks for her last requests. She forgives Elisabetta and prays for a blessing on her and the kingdom. She tries to calm the grief-stricken Leicester and hopes that her innocent blood will placate the wrath of Heaven. She goes resolutely to her death as her friends grieve over her fate.
