The Buryja mill. Dusk in September.

Jenůfa is waiting anxiously for Števa to return from the draft board. If he is conscripted she and Števa will not be able to get married, and her pregnancy will be discovered. She could face the rough justice of the village in the form of death by stoning, as well as the damnation of her soul, which her religious upbringing makes her fear. Laca, who has loved Jenůfa since childhood, jealously watches her. He resents his subservient position in the mill, and Jenůfa 's love for Števa. The mill girl Jana thanks Jenůfa for teaching her how to read. The mill foreman brings the news that Števa has not been recruited after all. Števa arrives drunk and accompanied by a band and the men who have been drafted. They dance and sing with the mill workers but their merrymaking is cut short by the Kostelnička who sees in Števa a husband as unsuitable as her own was: drunken, spendthrift and violent. Not knowing that Jenůfa is pregnant, the Kostelnička refuses to consent to their marriage unless they delay it for a year, during which Števa is to give up drinking. Jenůfa vainly tries to make Števa realise his obligations. Laca returns to taunt Jenůfa and slashes her cheek with his knife.

Jenůfa has told her stepmother about her pregnancy. To save them both from disgrace, the Kostelnička has hidden her away in her house and told everyone that Jenůfa has gone to Vienna. Her baby boy, also called Števa , is now eight days old.

The Kostelnička drugs Jenůfa 's drink. While Jenufa sleeps, Števa comes in answer to the Kostelnička 's summons. He agrees to pay for the upkeep of his baby but wants his fatherhood kept secret. He also refuses to marry Jenůfa, and announces he is now betrothed to Karolka, the Mayor's daughter. He runs away when he hears cry Jenůfa out in a nightmare.

Laca is more sympathetic, and still hopes to marry Jenůfa. But he is appalled when the Kostelnička tells him about the baby. On impulse, the Kostelnička tells him that it died. Left alone, she decides to save Jenůfa and herself from shame by killing the baby.

Jenůfa awakes and searches for the baby. She imagines her stepmother has taken little Števa to the mill, and prays to the Virgin to protect him. The Kostelnička returns and tells Jenůfa that she has been in a fever for two days, during which time the baby has died.
Laca returns and Jenůfa agrees to marry him if he still wants her. The Kostelnička blesses the couple and curses Števa, the cause of this misfortune. Suddenly the windows are blown open, and to the Kostelnička it seems like the voice of death forcing its way in.