The second act opens in the public square of Ferrara, with the palace of the Borgias on the right.  The Duke Alphonso, Lucrezia’s husband, who has been observant of Lucrezia’s attachment to Genarro, vows vengeance in a passionate aria ("Vieni la mia vendetta").  In the next scene Genarro, who has been taunted by his friends with being a victim of Lucrezia’s fascinations, recklessly rushes up to the palace door and strikes off the first letter of her name with his dagger.  When Lucrezia discovers the insult, she demands of the Duke that the guilty person shall be arrested and condemned to death.  The Duke has already seized Genarro, and agrees to carry out his wife’s demands.  When the prisoner is brought before them for judgment, she is horror-stricken to find he is her son.  She implores his life, but the infuriated Duke retaliates upon her with the declaration that she is his paramour.  The duet between them ("O! a te bada"), in which Lucrezia passes from humble entreaties to rage and menace, is a fine instance of Donizetti’s dramatic power.  The Duke, however, is resolute in his determination, and will only allow her to choose the mode of Genarro’s death.  She selects the Borgia wine, which is poisoned.  Genarro is called in, and after a trio ("Le ti tradisce"), which is one of the strongest numbers in the opera, he is given the fatal draught under the pretence of a farewell greeting from the Duke, who then leaves mother and son together.  She gives him an antidote, and he is thus saved from the fate which the Duke had intended for him.

The last act opens at a banquet in the palace of the Princess Negroni, which is attended by Genarro and his friends, Lucrezia, meanwhile, supposing that he has gone to Venice.  During the repast she has managed to poison their wine.  In the midst of the gay revel Orsini sings the popular drinking-song, “Il segreto per esser felici,” which is now familiar the world over.  The festivities are interrupted, however, by the appearance of Lucrezia, who reveals herself with the taunting declaration:  “Yes, I am Borgia.  A mournful dance ye gave me in Venice, and I return ye a supper in Ferrara.”  She then announces that they are poisoned.  The music is changed with great skill from the wild revelry of drinking-songs to the sombre strains of approaching death.  Five coffins are shown them, when Genarro suddenly reveals himself to Lucrezia and asks for the sixth.  The horror-stricken woman again perceives that her son has been poisoned by her own hand.  As his companions leave the apartment she implores Genarro to take the antidote once more, and at last reveals herself as his mother.  He steadily refuses to save himself, however, since his companions have to die, and expires in her arms just as the Duke and his followers enter.  She discloses Genarro’s relationship, and then dies with the despairing cry on her lips that Heaven has pronounced its final judgment upon her.  Among all of Donizetti’s operas, not one, unless it be “Lucia,” is more popular than “Lucrezia Borgia,” which may be attributed to the fact that while the story itself is one of fascinating dramatic interest, the musical numbers are simple, beautiful, and effective.