On a moonlit meadow near a lake, three Wood Nymphs sing, dance, and tease the water gnome Vodník, ruler of the lake's underwater realm, who pretends to try to catch them. As they leave, Vodník becomes aware of his daughter Rusalka, a water nymph, who tells her father of her love for the Prince, a mortal whom she often sees by the lake. She wishes to become human and have a soul. Horrified, the wise Vodník gloomily tells his daughter of the consequences of becoming human and advises her to consult the witch Ježibaba. Alone, Rusalka confides to the moon the secret of her longing and calls to Ježibaba, begging for her help. The witch tells Rusalka that the price of becoming human is very high: Rusalka must remain mute, and if she does not succeed in keeping the Prince's love, she must return as a spirit to the waters, where she will lure her love to his death. Heedless of the witch's warning, Rusalka insists, and Ježibaba brews a potion and gives it to Rusalka to drink. As dawn breaks, the Prince appears leading a party of hunters. They have been pursuing a white doe that has mysteriously vanished. The Prince senses something strange about this place and sends his hunters away, hoping to be alone with his thoughts. He meets Rusalka, who has been transformed into a lovely maiden, and is enchanted by her beauty. Unable to speak, Rusalka throws herself into the Prince's arms. He leads her off to his castle as the water nymphs and her father mourn her love.
At the castle, the Gamekeeper and the Kitchen Boy gossip about the upcoming marriage of the Prince to the strange, speechless maiden. They suspect witchcraft and are glad that the Prince's interest in the nameless bride is waning. He is already paying attention to one of the wedding guests, a beautiful Foreign Princess. The Prince enters with Rusalka. He wonders why she is so cold and shows no signs of love for him. When the Princess seductively talks to the Prince, Rusalka realizes the threat to her happiness and speaks to her father of her fear. The water gnome advises her to persevere, but Rusalka is no match for the aggressive Princess, who soon has enticed the fickle Prince with her charms. As the Prince rejects Rusalka, Vodník reappears, seizes his daughter and drags her away. The frightened Prince appeals to the Princess who cruelly laughs at him and departs.
Rusalka sits alone by the moonlit lake, speaking of her sorrow. Ježibaba emerges from her cottage and tells the sad young creature that in order to wash away the curse, she must now kill the Prince. Rusalka is unwilling to consider this but is resigned to her fate. As the water nymphs comment on her short stay in the human world, Rusalka slowly vanishes.
Frightened, the Gamekeeper and the Kitchen Boy have come to the woods to ask Ježibaba for advice about the Prince, who has fallen ill, cursed by some strange spell. Vodník appears and chases the timid pair away, cursing the human race.
The Prince emerges from the forest like a madman, crying out for Rusalka and calling upon heaven and earth to help him find her. Appearing as a spirit in the moonlight above the lake, Rusalka speaks to the Prince, who rushes into her arms. Rusalka kisses him and he dies as he begs heaven to be merciful to him.