During Carnival celebrations before Lent, while everyone else is preoccupied with a regatta, Barnaba, a state spy, lustfully watches La Gioconda as she leads her blind mother, La Cieca, across the Square. When his amorous advances are firmly rejected, he exacts his revenge by denouncing the old lady as a witch whose evil powers influenced the outcome of the gondola race. It is only the intervention of a young sea captain that keeps the angry mob at bay.
Calm is restored at the approach of Alvise Badoero, a member of the Venetian Inquisition, and his wife, Laura. Laura places La Cieca under her personal protection, and in gratitude the old woman presents her with her most treasured possession, a rosary. The sharp-eyed Barnaba notices furtive behaviour between Laura and the sea captain indicating a secret relationship. Recalling that Laura was engaged to the now banished nobleman Enzo Grimaldo before her forced marriage to Alvise, Barnaba realises that the sea captain is Enzo in disguise. Barnaba confronts Enzo, who admits his purpose in returning to Venice is to take Laura and begin a new life elsewhere. Barnaba knows that Gioconda is also infatuated with Enzo and he sees an opportunity to improve his chances with her by assisting Enzo with his plan of elopement. When Enzo has gone, Barnaba dictates a letter to Alvise revealing his wife's infidelity and the lovers' plan of escape. He is unaware that he has been overheard by Gioconda. The act ends with Barnaba dropping the letter into the Lion's Mouth, where all secret information for the Inquisition is posted, while Gioconda laments Enzo's perceived treachery, and the crowd returns to its festivities.
At the deck of Enzo's ship, Enzo waits for Barnaba to row Laura out from the city to his vessel. Their joyful reunion is overshadowed by Laura's fears as she does not trust Barnaba. Gradually Enzo is able to reassure her, and he leaves her on deck while he goes to prepare for their departure. La Gioconda has been following Laura with the intention of exacting revenge from her rival. Alvise and his armed men are also in hot pursuit, but as Gioconda is about to stab Laura she sees her mother's rosary hanging round her neck and has an instant change of heart. She hurries Laura into her boat so that she can evade her pursuers. Enzo returns to the deck to find that Laura has fled leaving Gioconda triumphant. Furthermore, Alvise's men are rapidly approaching. He sets fire to the ship rather than let it fall into the hands of his enemies before diving into the lagoon.
At the Ca' d'Oro, Alvise's palace, Laura has been captured, and her vengeful husband insists she must die by poisoning herself (effectively committing suicide and condemning herself to Hell). Once again Gioconda has followed and has found her way into the palace, this time with the intention of saving her rival. Finding Laura alone Gioconda replaces the phial of poison with a powerful drug which creates the appearance of death. The second scene begins with Alvise welcoming his fellow members of the nobility to the palace; Barnaba and Enzo are amongst those present. Lavish entertainment is provided and the act ends with the famous ballet Dance of the Hours. The mood of revelry is shattered as a funeral bell begins to toll and the body of Laura is revealed awaiting burial. A distraught Enzo flings off his disguise and is promptly seized by Alvise's men.
A crumbling ruin on the island of Giudecca. In exchange for Enzo's release from prison, La Gioconda has agreed to give herself to Barnaba. When Enzo is brought in, he is initially furious when Gioconda reveals that she has had Laura's body brought from its tomb. He is about to stab her when Laura's voice is heard and Gioconda's part in reuniting the lovers becomes clear. Enzo and Laura make their escape, leaving La Gioconda to face the horrors awaiting her with Barnaba. The gondoliers' voices are heard in the distance telling that there are corpses floating in the city. When Gioconda tries to leave, she is caught by Barnaba. She then pretends to welcome his arrival, but under cover of decking herself in her jewellery, seizes a dagger and stabs herself to death. In frustrated rage Barnaba tries to perpetrate one last act of evil, screaming at the lifeless body that the night before her mother ofended him and he drowned her.
