The play consists of four interconnecting plots, connected by a celebration of the wedding of Duke Theseus of Athens and the Amazon queen, Hippolyta, which is set simultaneously in the woodland and in the realm of Fairyland, under the light of the moon.
The play opens with Hermia, who is in love with Lysander, not wanting to submit to her father Egeus' demand that she wed Demetrius, whom he has arranged for her to marry. Helena meanwhile pines unrequitedly for Demetrius. Enraged, Egeus invokes an ancient Athenian law before Duke Theseus, whereby a daughter must marry the suitor chosen by her father, or else face death. Theseus offers her another choice: lifelong chastity while worshiping the goddess Diana as a nun.
Peter Quince and his fellow players plan to put on a play for the wedding of the Duke and the Queen, "the most lamentable comedy and most cruel death of Pyramus and Thisbe". Quince reads the names of characters and bestows them to the players. Nick Bottom, who is playing the main role of Pyramus, is over-enthusiastic and wants to dominate others by suggesting himself for the characters of Thisbe, the Lion, and Pyramus at the same time. He would also rather be a tyrant and recites some lines of Ercles. Quince ends the meeting with "at the Duke's oak we meet".
In a parallel plot line, Oberon, king of the fairies, and Titania, his queen, have come to the forest outside Athens. Titania tells Oberon that she plans to stay there until she has attended Theseus and Hippolyta's wedding. Oberon and Titania are estranged because Titania refuses to give her Indian changeling to Oberon for use as his "knight" or "henchman," since the child's mother was one of Titania's worshipers. Oberon seeks to punish Titania's disobedience. He calls upon Robin "Puck" Goodfellow, his "shrewd and knavish sprite", to help him concoct a magical juice derived from a flower called "love-in-idleness", which turns from white to purple when struck by Cupid's arrow. When the concoction is applied to the eyelids of a sleeping person, that person, upon waking, falls in love with the first living thing they perceive. He instructs Puck to retrieve the flower with the hope that he might make Titania fall in love with an animal of the forest and thereby shame her into giving up the little Indian boy. He says, "And ere I take this charm from off her sight, / As I can take it with another herb, / I'll make her render up her page to me."
Hermia and Lysander have escaped to the same forest in hopes of eloping. Helena, desperate to reclaim Demetrius's love, tells Demetrius about the plan and he follows them in hopes of killing Lysander. Helena continually makes advances towards Demetrius, promising to love him more than Hermia. However, he rebuffs her with cruel insults against her. Observing this, Oberon orders Puck to spread some of the magical juice from the flower on the eyelids of the young Athenian man. Instead, Puck mistakes Lysander for Demetrius, not having actually seen either before, and administers the juice to the sleeping Lysander. Helena, coming across him, wakes him while attempting to determine whether he is dead or asleep. Upon this happening, Lysander immediately falls in love with Helena. Oberon sees Demetrius still following Hermia and is enraged. When Demetrius decides to go to sleep, Oberon sends Puck to get Helena while he charms Demetrius' eyes. Upon waking up, he sees Helena. Now, both men are in pursuit of Helena. However, she is convinced that her two suitors are mocking her, as neither loved her originally. Hermia is at a loss to see why her lover has abandoned her, and accuses Helena of stealing Lysander away from her. The four quarrel with each other until Lysander and Demetrius become so enraged that they seek a place to duel each other to prove whose love for Helena is the greatest. Oberon orders Puck to keep Lysander and Demetrius from catching up with one another and to remove the charm from Lysander. Lysander returns to loving Hermia, while Demetrius continues to love Helena.
Meanwhile, Quince and his band of six labourers ("rude mechanicals", as they are described by Puck) have arranged to perform their play about Pyramus and Thisbe for Theseus' wedding and venture into the forest, near Titania's bower, for their rehearsal. Bottom is spotted by Puck, who (taking his name to be another word for a jackass) transforms his head into that of a donkey. When Bottom returns for his next lines, the other workmen run screaming in terror, much to Bottom's confusion, since he hasn't felt a thing during the transformation. Determined to wait for his friends, he begins to sing to himself. Titania is awakened by Bottom's singing and immediately falls in love with him. She lavishes him with attention and presumably makes love to him. While she is in this state of devotion, Oberon takes the changeling. Having achieved his goals, Oberon releases Titania, orders Puck to remove the donkey's head from Bottom, and arranges everything so that Hermia, Lysander, Demetrius, and Helena will believe that they have been dreaming when they awaken.
The fairies then disappear, and Theseus and Hippolyta arrive on the scene, during an early morning hunt. They wake the lovers and, since Demetrius does not love Hermia any more, Theseus overrules Egeus's demands and arranges a group wedding. The lovers decide that the night's events must have been a dream. After they all exit, Bottom awakes, and he too decides that he must have experienced a dream "past the wit of man". In Athens, Theseus, Hippolyta and the lovers watch the six workmen perform Pyramus and Thisbe. Given a lack of preparation, the performers are so terrible playing their roles to the point where the guests laugh as if it were meant to be a comedy, and everyone retires to bed. Afterwards, Oberon, Titania, Puck, and other fairies enter, and bless the house and its occupants with good fortune. After all other characters leave, Puck "restores amends" and suggests to the audience that what they just experienced might be nothing but a dream (hence the name of the play).