Gennaro leaves his friends and falls asleep on a terrace. He is discovered by Lucrezia Borgia, who is masked. He wakes and, charmed by her concern, tells her of his life and how he was raised by a lowly fisherman. He is horrified, however, when his friends tear off her mask and reveal that she is the infamous Lucrezia Borgia.
Lucrezia's fourth husband Alfonso suspects her of infidelity but she is only seeing Gennaro - who is in fact her son by a previous marriage. Gennaro is ignorant of his mother's identity and, keen to show his hatred of Lucrezia's crimes, he cuts off the first letter of her name from the palace gates, leaving only "orgia" (orgy).
Enraged by this insult, Lucrezia demands revenge. Alfonso brings in Gennaro and forces him to take poison but, realizing the identity of the culprit, Lucrezia administers an antidote and begs him to leave the city.
At a banquet in Ferrara, Gennaro and his friends mock Lucrezia. She appears and informs them that they have all drunk poisoned wine, but when she sees her son she clears the hall and offers him an antidote. Revolted by her actions and shattered by her claim that she is his mother, he refuses the potion and dies. Crushed by his rejection, she drains the poisoned cup.