A garden in Serebryakov's country estate. Astrov and Marina discuss how old Astrov has grown, and how he feels bored with his life as a country doctor. Vanya enters, yawning from a nap, the three complain about how all order has been disrupted since the professor and his wife, Yelena, arrived. As they’re talking, Serebryakov, Yelena, Sonya, and Telegin return from a walk. Out of the professor's earshot, Vanya calls him "a learned old dried mackerel," criticizing him for his pomposity and the smallness of his achievements. Vanya’s mother, Maria Vasilyevna, who idolizes Serebryakov, objects to her son’s derogatory comments. Vanya also praises the professor’s wife, Yelena, for her beauty, arguing that faithfulness to an old man like Serebryakov means silencing youth and emotions — an immoral waste of vitality. Astrov is forced to depart to attend a patient, but not before delivering a speech on the preservation of the forests, a subject he is very passionate about. Act I closes with Yelena becoming exasperated as Vanya declares his love for her.
The dining room, several days later. It is late at night. Before going to bed, Serebryakov complains of being in pain and of old age. Astrov arrives, having been sent for by Sonya, but the professor refuses to see him. After Serebryakov is asleep, Yelena and Vanya talk. She speaks of the discord in the house, and Vanya speaks of dashed hopes. He feels he’s misspent his youth, and he associates his unrequited love for Yelena with the devastation of his life. Yelena refuses to listen. Alone, Vanya questions why he did not fall in love with Yelena when he first met her ten years before, when it would have been possible for the two to have married and had a happy life together. At that time, Vanya believed in Prof. Serebryakov’s greatness and was happy to think that his own efforts supported Serebryakov's work; now he has become disillusioned with the professor and his life feels empty. As Vanya agonizes over his past, Astrov returns, the worse for drink, and the two talk together. Sonya chides Vanya for his drinking, and responds pragmatically to his reflections on the futility of a wasted life, pointing out that only work is truly fulfilling.
Outside, a storm is gathering and Astrov talks with Sonya about the suffocating atmosphere in the house; Astrov says Serebryakov is difficult, Vanya is a hypochondriac, and Yelena is charming but idle. He laments that it’s a long time since he loved anyone. Sonya begs Astrov to stop drinking, telling him it is unworthy of him to destroy himself. The two discuss love, during which it becomes clear that Sonya is in love with the Doctor and that he is unaware of her feelings.
When the doctor leaves, Yelena enters and makes peace with Sonya, after an apparently long period of mutual anger and antagonism. Trying to resolve their past difficulties, Yelena reassures Sonya that she had strong feelings for her father when she married him, though the love proved false. The two women converse at cross purposes, with Yelena confessing her unhappiness and Sonya gushing about the doctor’s virtues. In a happy mood, Sonya leaves to ask the professor if Yelena may play the piano. Sonya returns with his negative answer, which quickly dampens the mood.
Vanya, Sonya, and Yelena are in the living room, having been called there by Serebryakov. Vanya calls Yelena a water nymph and urges her, once again, to break free. Sonya complains to Yelena that she has loved Astrov for six years but that, because she is not beautiful, he doesn’t notice her. Yelena volunteers to question Astrov and find out if he’s in love with Sonya. Sonya is pleased, but before agreeing she wonders whether uncertainty is better than knowledge, because then, at least, there is hope.
When Yelena asks Astrov about his feelings for Sonya, he says he has none and concludes that Yelena has brought up the subject of love to encourage him to confess his own emotions for her. Astrov kisses Yelena, and Vanya witnesses the embrace. Upset, Yelena begs Vanya to use his influence so that she and the professor can leave immediately. Before Serebryakov can make his announcement, Yelena conveys to Sonya the message that Astrov doesn’t love her.
Serebryakov proposes that he solve the family’s financial problems by selling the estate, and using the proceeds to invest in interest-bearing paper which will bring in a significantly higher income (and, he hopes, leave enough over to buy a villa for himself and Yelena in Finland). Angrily, Vanya asks where he, Sonya, and his mother would live. He protests that the estate rightly belongs to Sonya and that Vanya has never been appreciated for the self-sacrifice it took to rid the property of debt. As Vanya’s anger mounts, he begins to rage against the professor, blaming him for the failure of his life, wildly claiming that, without Serebryakov to hold him back, he could have been a second Schopenhauer or Dostoevsky. In despair, he cries out to his mother, but instead of comforting her son, Maria insists that Vanya listen to the professor. Serebryakov insults Vanya, who storms out of the room. Yelena begs to be taken away from the country and Sonya pleads with her father on Vanya's behalf. Serebryakov exits to confront Vanya further. A shot is heard from offstage and Serebryakov returns, being chased by Vanya, who is wielding a loaded pistol. He fires the pistol again at the professor, but misses. He throws the gun down in disgust and sinks into a chair.
As the final act opens, a few hours later, Marina and Telegin wind wool and discuss the planned departure of Serebryakov and Yelena. When Vanya and Astrov enter, Astrov says that in this district only he and Vanya were "decent, cultured men" and that ten years of "narrow-minded life" have made them vulgar. Vanya has stolen a vial of Astrov’s morphine, presumably to commit suicide; Sonya and Astrov beg him to return the narcotic, which he eventually does.
Yelena and Serebryakov bid everyone farewell. When Yelena says goodbye to Astrov, she admits to having been carried away by him, embraces him, and takes one of his pencils as a souvenir. Serebryakov and Vanya make their peace, agreeing all will be as it was before. Once the outsiders have departed, Sonya and Vanya pay bills, Maria reads a pamphlet, and Marina knits. Vanya complains of the heaviness of his heart, and Sonya, in response, speaks of living, working, and the rewards of the afterlife: "We shall hear the angels, we shall see the whole sky all diamonds, we shall see how all earthly evil, all our sufferings, are drowned in the mercy that will fill the whole world. And our life will grow peaceful, tender, sweet as a caress. . . . You've had no joy in your life; but wait, Uncle Vanya, wait. . . . We shall rest."