The principal figures in "Jenufa" represent three generations of one family. Grandmother Buryja had two sons, the elder of whom married the widow Klemen, who already had one son, Laca. They had one son Steva, who inherited the family mill.
The second son, widowed, with a daughter, Jenufa, married the Kostelnicka (female sacristan, so called because she tended the village church) as his second wife, and she brought up Jenufa as her own child.
The courtyard of the mill, Jenufa anxiously watches for the arrival of her sweetheart Steva, fearing that he may be conscripted and their wedding postponed, which would bring her shame as she is pregnant. When her grandmother reproves her for not attending to her work, Laca (who is watching Jenufa) reproaches her for neglecting him as a child, in favor of his half-brother, Steva. Jenufa, who fears he has detected her secret, speaks sharply to him. Jano the shepherd boy runs in announcing that Jenufa has taught him to read.
Asking the mill foreman to sharpen his knife, Laca teases Jenufa and when she turns on him, he remarks what a fine sister-in-law she will make, but the foreman is not deceived about the nature of Laca's feelings. Laca's hopes that Steva has been conscripted are dashed - the foreman has just learnt that Steva has not been taken. Steva appears with other lads from the village, accompanied by musicians.
Jenufa is upset because he is drunk, but he orders the musicians to play her favorite song and leads everyone into a dance, which comes to an abrupt halt when the Kostlenicka appears. She tells Steva that he is not yet fit to marry Jenufa - he must go for a year without getting drunk before she will consider it. Jenufa tries to plead with her, but she answers that God will punish her if she disobeys. Only Laca is delighted and kisses her hand.
Jenufa begs Steva not to antagonise her stepmother, as she will be shamed if the wedding has to be put off. His idea of comforting her is to tell her how much all the girls admire him, but to him she is the prettiest of all, with her cheeks like rosy apples.
The grandmother calls him inside and Laca again comes up to Jenufa. They quarrel and his knife accidentally slashes her cheek. He cries out in remorse that he has loved her all his life, while the foreman accuses him of having done it on purpose.
Inside the Kostelnicka's house, some months later Jenufa has had her baby, unknown to the village, as the Kostelnicka has kept her indoors and given out that she has gone to Vienna. Sending Jenufa to bed with a sleeping draught, the Kostelnicka prepares to receive Steva, who has not been near Jenufa since her accident and still does not know about the birth of the baby. Although she hates him, she is prepared to humble herself for Jenufa's sake.
But Steva refuses to aknowledge the baby, though he is willing to support it. Despite the entreaty of the Kostelnicka, he refuses to marry Jenufa, saying she has lost her beauty and become as witch-like as the Kostelnicka, whom he fears. Besides, he is going to marry the mayor's daughter, Karolka.
His departure is followed immediately by the arrival of Laca, who has been a constant visitor to the Kostelnicka, though unaware of Jenufa's presence and of the existence of the baby. Although he wants to marry Jenufa, he is appalled to learn about the child, so the Kostelnicka tells him it has died. Sending him on an errand, she takes the baby to the icy river.
Jenufa awakes and misses the baby, a boy called Steva. She calms her fears by telling herself that the Kostelnicka must have taken him to the mill to show everyone and comforts herself with the thought that Steva will now come to see her and the baby. She prays for the child. The Kostelnicka comes back without the baby and tells Jenufa that she has been in a fever for days and that the baby died. She also tells her that Steva refused to have anything to do with her or the baby, and advises her to marry Laca, a man she can trust. He knows everything and has forgiven her.
He returns and Jenufa yields to his entreaties and the urgings of the Kostelnicka and agrees to marry him. The Kostelnicka blesses them and curses Steva. The window blows open and she cries that the icy hand of death is forcing its way in.
As in Act II, some months later. It is spring and Jenufa and Laca are about to be married. Although she is pleased, the Kostelnicka's health is weakened by her burden of guilt.
Jenufa tells Laca he deserves a better bride, but he repeats that he has forgiven her and reminds her of the wrong he did her, for which he intends to spend his life atoning. He tells her he is now reconciled with Steva, who is coming to the wedding with Karolka. When they arrive Jenufa gets the brothers to shake hands and tells Steva she is glad he has found true love.
The village girls sing a wedding song and the grandmother blesses Jenufa and Laca. Jano rushes in with the news that a baby's body has been found under the melting ice in the river. Jenufa recognises the clothes, but cannot understand how her baby was found in the river. The villagers think she has killed her child and are ready to stone her, but Laca defends her furiously. The Kostelnicka confesses, explaining that she committed the murder to save Jenufa from shame. Jenufa shrinks from her in horror. Realising that Steva is the father of the baby, Karolka refuses to marry him.
The Kostelnicka now realises that she had been thinking more of herself than of Jenufa, but Jenufa finds the strength to forgive her as she is led away by the mayor.
Jenufa offers to relase Laca, but he refuses to leave her and she realises that she now truly loves him. They prepare to face the future together.