In a room in the Garter Inn, Sir John Falstaff is writing letters. Dr. Caius enters and complains of the various outrages that have been committed against him by the knight and his followers, Pistola and Bardolfo. Falstaff laughs off these accusations and Dr. Caius storms out of the tavern. Finding they have no money to pay the bill, Falstaff blames his companions and announces a plan to repair his fortunes by having affairs with Alice Ford and Meg Page, both of whom are married to wealthy men ("V'è noto un tal"). He produces the two love letters and tells Pistola and Bardolfo to deliver one to each woman, but the men refuse to assist in such a dishonorable enterprise. Falstaff sends the letters off with a page instead and then lectures the two men on the subject of honor ("L'onore! Ladri!") before chasing them out of the inn.

In the garden outside of Ford's house, Alice Ford and her daughter Nannetta meet with Meg Page and Dame Quickly. Alice and Meg are each bursting with the news of their letter from Falstaff. Upon comparing the letters they find them to be identical and the women decide he must be taught a lesson ("Fulgida Alice! Amor t'offro"). Meanwhile, Ford meets with Dr. Caius, Bardolfo, Pistola, and young Fenton, who is secretly in love with Nannetta. Bardolfo and Pistola have betrayed their master and told Ford that Falstaff is pursuing his wife. Briefly alone, Fenton and Nannetta steal kisses but are interrupted when the women return, plotting to send Dame Quickly to Falstaff to arrange an assignation with Alice. Nannetta and Fenton are next interrupted by Ford, who is also plotting his revenge, and plans to be introduced to Falstaff as 'Master Brook,' so that he can keep an eye on him. The women return once more and, unaware of each other's plans, all pledge to punish the knight.

Back at the Garter Inn, Falstaff accepts the feigned apologies of Bardolfo and Pistola. Dame Quickly arrives to tell the knight that both Alice and Meg return his affections. She says that Meg's husband guards her too closely to ever leave her alone but that Alice will meet with him between two and three that afternoon when her husband is always out. Once the meeting is set, Dame Quickly leaves and Falstaff begins to preen himself in preparation for his rendezvous. Ford enters the inn and introduces himself to Falstaff as 'Master Brook,' a wealthy man who has fallen in love with Alice Ford but has been unable to woo her. He offers Falstaff money for his help in the matter, suggesting that if the knight can break down Alice's resistance, he will be able to follow suit. Falstaff accepts the challenge, gleefully boasting that he himself has already set up a tryst with the lady and excuses himself to get ready. Left alone, Ford can't believe his wife's betrayal and vows to avenge his honor ("È sogno? o realtà"). He regains his composure by the time Falstaff returns, and the two men leave together.

Back at Ford's house, Dame Quickly gives a detailed report of her meeting with Falstaff. Nannetta comes in and tearfully tells them that Ford has ordered her to marry old Dr. Caius. When she says that she is in love with Fenton and will marry no one but him, her mother promises to help her. The other women hide and Alice sits at her lute as Falstaff arrives. She listens to his advances but keeps the knight at a safe distance. They are interrupted by Dame Quickly, who tells them that Ford is on his way. Falstaff hides behind a screen just as Ford enters with his followers, hoping to catch the knight seducing his wife. Finding nothing, the men leave to search the rest of the house. While Ford is searching the other rooms, the women hide Falstaff in a laundry basket, covering him with dirty linens. Ford returns and, hearing the sound of kissing from behind a screen, discovers Nannetta and Fenton. More furious than ever, Ford rushes out to continue his search, while Alice orders the servants to dump the laundry basket out the window and into the Thames River below. When Ford returns, Alice leads him to the window just in time to see Falstaff dumped into the muddy river.

Back at the inn, Falstaff broods over his humiliation, but his spirits improve drastically as he drinks his wine ("Mondo ladro"). Dame Quickly comes with a message from Alice, but Falstaff wants nothing more to do with her. She persuades him that it was the servants' fault that he ended up in the river and that Alice wants to meet with him again. Falstaff cannot resist the temptation and agrees to a midnight meeting in Windsor Park, where he is to come disguised as the 'Black Huntsman,' who is said to haunt the forest. The women plan the details of the midnight masquerade. Fenton and Caius participate in the plans as well, along with Ford, who is now convinced of his wife's fidelity. Dame Quickly reappears to overhear Ford and Caius plotting to announce Nannetta's marriage to the doctor that very night, and she vows to stop them.

That night in Windsor Forest, Fenton sings of his love for Nannetta ("Dal labbro il canto") but is interrupted by Alice, who gives him a costume identical to the one that Ford has given Caius to wear. The rest of the group hastily dons their costumes and hides just as Falstaff arrives dressed as the Black Hunter. Alice greets him, but when they hear noises, she quickly disappears, leaving him alone as a horde of spirits descends on him. Nannetta, who is disguised as Queen of the Fairies, calls to her followers, who gather around the knight as he cowers on the ground ('Sul fil d'un soffio etesio"). Begging for mercy, Falstaff finally recognizes Bardolfo through his disguise. The rest of the group unmasks and Falstaff realizes that he has been played for a fool. The ladies reprove him for thinking that they would have accepted him as a lover and Ford reveals himself as Alice's husband, not the lovelorn 'Brook.' Ford takes Nannetta by the hand and announces her betrothal to Caius. He does the same for a similarly disguised couple brought forward by Alice. He bids them all unmask and is shocked to find that Bardolfo has been dressed up in Nannetta's clothes and is now therefore betrothed to Caius. The other couple is, of course, Nannetta and Fenton in disguise. Ford has been duped too but he forgives them all, and Falstaff leads the group in declaring that the world is but a jest ("Tutto nel mondo è burla").