Caesar Angelotti, an escaped political prisoner, runs into the church of Sant'Andrea della Valle to hide in the Attavanti family chapel. At the sound of the Angelus, the Sacristan enters to pray. He is interrupted by Mario Cavaradossi, who has come to work on his portrait of Mary Magdalene - inspired by the Marchesa Attavanti, Angelotti's sister. Mario contrasts the beauty of the blond Marchesa with that of the woman he loves, the raven-haired singer Floria Tosca ("Recondita armonia.")

Angelotti ventures out and is recognized by Mario, who gives him food and hurries him back into the chapel as Tosca is heard calling outside. She jealously questions Mario, then prays and reminds him of their rendezvous that evening ("Non las sospiri la nostara casetta?") Recognizing the Marchesa's likeness in the painting, she explodes with renewed suspicions, but he reassures her ("Qual'occhio al mondo.") When she has left, Mario summons Angelotti as a cannon signals that the police have discovered the escape; the two flee to Mario's villa.

The Sacristan returns with choirboys who are about to sing a Te Deum. Their excitement is silenced by the entrance of Baron Scarpia, chief of the secret police, in search of Angelotti. When Tosca comes back looking for Mario, she encounters Scarpia, who shows her the Attavanti crest on a fan he has just found. Thinking Mario faithless, Tosca tearfully vows vengeance and leaves as the church resounds with the Te Deum. Scarpia has the diva trailed, scheming to get her in his power ("Va, Tosca!")