Grandmother Buryja had two sons. One of them married a widow who had a son – Laca Klemeň – from her earlier marriage. Števa was born from the later marriage. Jenůfa is the daughter of the other Buryja son. After the death of this other son's wife, Jenůfa's mother, he married another woman, and this young Buryja became Jenůfa's stepmother. Her husband drank himself to death, forcing her to run a market stall to support the two of them. Later she was asked to fill the vacant position of church sacristan, which is how she took the name Kostelnička. Laca and Števa have both fallen in love with Jenůfa, but it is Števa's feelings that the girl reciprocates. Laca jealousy toward his half-brother is compounded by the fact that the Buryja family's mill that had become his mother's property after his stepfather died has been inherited by Števa, as he was Buryja's natural son from the marriage, and Laca has to work there as an apprentice. Living at the mill is Grandmother (to Števa and Jenůfa, step-grandmother to Laca) Buryja, where she is not an owner, but merely receives room and board, her sense of prestige worn down by the fact that she plays a formal role in family rituals, but is otherwise not respected. Jenůfa also works at the mill and helps her grandmother with the housework.
Afternoon in front of the mill. Jenůfa anxiously awaits Števa's return from appearing for conscription. If he has to enlist, they will not be able to be married, and she is expecting his child. Laca is also in love with the girl, but she does not reciprocate his emotions; Grandmother Buryja scolds the boy for not helping her with the housework. The young shepherd boy Jano jubilantly thanks Jenůfa for teaching him how to read. Laca remains alone with the old mill foreman and tells him that he hopes that Števa will be drafted, but the older man informs him and the gathering family members that the lad will not have to enlist after all. The latter then appears, together with new recruits and musicians, and launches into such wild revelry that Kostelnička Buryja, Jenůfa's step-mother, forbids them from marrying until he has served a year of probation: Števa must prove through his conduct that he is worthy of the girl. The villagers and family members disperse. Števa reassures Jenůfa and departs. Laca again attempts to approach the girl, but she she rejects him. The jealous young man slashes Jen?fa's face in order to disfigure it, but immediately regrets his deed.
Six months later, a cold winter's night in Kostelnička's house. This is where the step-mother has hidden Jenůfa, who gave birth to a child, little Števa, eight days earlier. Expecting Števa to come, Kostelnička mixes a sedative into the girl's drink to send her to sleep. She attempts to persuade the young man to marry Jenůfa, but to no avail, and so she turns to Laca, who has remorsefully assured her that he still loves the girl. When he learns that Jenůfa has born a child, however, he is thrown into confusion, and Kostelnička suddenly lies to him that the child has died. She sends him out on the pretext of finding out whether there is any truth to the news that Števa has been engaged to Karolka, the daughter of the village judge. She decides right there: the child must die. She hurries out with him and throws him under the ice in the brook. Jenůfa awakens from her troubled sleep and, unable to find her infant son, starts to pray to the Virgin Mother. Kostelnička returns and convinces her that she has had a high fever for two days, and the child died in the meanwhile. The submissive Jenůfa feels that this is the judgement of God and agrees to marry Laca, who has returned. Kostelnička is already overcome with guilt.
Two months later, in the same place. Preparations are underway for Jenůfa's and Laca's wedding. Kostelnička has been acting troubled ever since the murder. The village's judge and his wife arrive with Števa, followed by a chorus of girls, which sings a bridal song to Jenůfa. Before the two proceed into the church, Grandmother Buryja blesses them. Then they ask for a blessing from Kostelnička, who starts to pull herself together when a sudden cry is heard from outside: in the melting ice of the brook, someone has found a dead child. Jenůfa recognises her little son from the shreds of clothes. Laca prevents the assembled villagers from taking revenge.
Kostelnička confesses her deed. Jenůfa is at first outraged, but when she realises that her step-mother had committed murder in order to help her, she forgives her. Kostelnička is led away. Karolka declares Števa the guilty party and calls off the engagement. Laca stands by Jenůfa and offers to leave the village with her. Jenůfa's love for Laca is rekindled and they face the future together.