After graduating from Bologna University, Giovanni returns to his home town of Parma. He has developed an incestuous passion for his sister, Annabella, which he discusses with Friar Bonaventura. The Friar tries hard to convince Giovanni that his desires for his sister come from a place of evil but Giovanni cannot comprehend that his feelings are anything but beautiful. However, he does agree to try to repent in an effort to put a stop to these feelings.
Meanwhile, his sister, Annabella, is being pursued by a number of suitors, including Grimaldi, Bergetto, and Soranzo, but she is not attracted to or interested in any of them. Giovanni decides to confess his feelings to her and she reciprocates (obviously his efforts to repent did not prove effective). Unbelievably their relationship is encouraged by Annabella's tutoress, Putana (whose name translates as "whore"), and they soon begin a sexual relationship.
Elsewhere, we learn that Saranzo has recently arranged for Richardetto, the husband of one of his ex-lovers, Hippolita, to undertake a very dangerous mission that will result in his death, so that Saranzo and Hippolita can rekindle their flame and finally be together. However, Hippolita is furious because although she kept her side of the bargain and sent Richardetto off on his journey, Saranzo seems to have gone back on his word and has abandoned her. Saranzo's servant Vasques promises Hippolita that he will help her avenge this humiliation and the pair agree that they will murder Saranzo. Richardetto, though, did not die on his journey as they had planned, and is living in Parma, also furious with Saranzo and plotting his revenge. He convinces Grimaldi that Saranzo is his main competition when it comes to winning Annabella's love and favor and also convinces him that he should stab Saranzo with a poisoned sword. The plan fails miserably because Bergetto and Philotis, who have now become engaged, arrange to marry secretly in exactly the place that Richardetto has sent Grimaldi to wait for Saranzo. Mistakenly, Grimaldi stabs Bergetto instead of Saranzo, which leaves Philotis distraught.
Annabella realizes that she cannot marry her brother despite their feelings for each other, so she decides to marry Saranzo. It is then revealed that she is pregnant, obviously by her brother. Friar Bonaventura urges her to marry quickly before her pregnancy becomes apparent to everyone. Their wedding ceremony includes masked dancers, one of whom turns out to be Hippolita. She toasts Saranzo and the two raise their glasses and drink, and at this moment she tells him that she has poisoned his wine. However, Vasques admits that his loyalty was always to his master and not to her, and he has actually poisoned her wine instead. As she dies, she curses the newlyweds. In some ways her curses seem to take immediate effect because when Saranzo finds out that Annabella is pregnant, he is devastated, and she realizes that the marriage was not for show at all, and that he genuinely loves her. Saranzo keeps her under lock and key in her bedroom and vows to find out who the father is so that he can avenge his humiliation. Vasques pretends to be very friendly towards Putana in an effort to get the name of Annabella's lover out of her. She reveals that it is Giovanni. He is so repulsed that he has her tied up and her eyes gouged out as a punishment for seeing these terrible acts and doing nothing to stop them.
Annabella writes Giovanni a letter in her own blood, telling him that Saranzo has found out that they were lovers and warning him that some kind of revenge is imminent. The Friar delivers the letter for her and warns Giovanni to keep his distance, and to decline the invitation he has received to attend Saranzo's birthday feast. True to form, Giovanni is too arrogant to accept advice and assumes that he knows best; he does not believe any harm will come to him and so accepts the celebration invitation. The Friar leaves Parma because he cannot bear to witness Giovanni's downfall, and feels that he has failed in his attempts to intervene in the situation.
Feast day dawns; Giovanni goes to Annabella's room to visit with her and stabs her in the heart with a dagger as they passionately kiss. He then goes to the feast, at which all of the surviving characters are present, with her heart still impaled on the tip of the dagger, and he tells everyone about their incestuous relationship. The shock of this news kills Florio, Annabella and Giovanni's father, immediately. Saranzo verbally berates Giovanni, but Giovanni stabs him too and kills him. Vasques attempts to kill Giovanni but only succeeds in wounding him, and so orders that bandits finish the job.
The Cardinal deals out swift punishment for the massacre at the feast. Putana is to be burned at the stake. Vasques is exiled. The Church lucks out; they are awarded all of the riches belonging to the dead. The play ends with the Cardinal observing about Annabella, "who could not say, 'tis a pity she's a whore?"
