Banquo and his son Fleance arrive at Inverness Castle late at night, commenting on the hour and the darkness. Macbeth enters with a servant bearing a torch; Banquo mentions Duncan’s generous gifts and recounts the witches’ prophecies. Macbeth and Banquo discuss the prophecies, and Banquo departs. Alone, Macbeth wrestles with a vivid hallucination of a floating dagger pointing toward Duncan’s chambers, which he interprets as a summons to murder. He hears a bell, sees the dagger's bloodied tip, and resolves to act, delivering the famous soliloquy about “sleep no more” and the darkness of his deed. He exits, presumably to kill Duncan.

In the next scene, Lady Macbeth, waiting in the castle, declares that the drunken grooms have been drugged and that she has placed the daggers ready for Macbeth. Macbeth returns, proclaiming he has “done the deed.” They converse anxiously about the noise, the king’s murder, and the reactions of the sleeping servants. Macbeth is tormented by visions of the murdered king’s voice urging “Sleep no more!” and is unable to pronounce “Amen.” Lady Macbeth urges him to wash the blood from his hands, to plant the daggers on the grooms, and to smear them with blood so they will be blamed. Macbeth refuses, fearing the sight of his own hands, and Lady Macbeth finally takes the daggers herself, mocking his childish fear.

A knocking is heard. Macbeth, increasingly paranoid, imagines the knocking as an accusation and wonders if the world will wash away his blood. Lady Macbeth reappears, insisting they must hide the crime, change into nightgowns, and dismiss the knocking. She laments the stain on their hands but urges Macbeth to proceed.

The Porter then enters, delivering comic relief with a series of “knocks” as if he were the gatekeeper of hell, before Macduff and Lennox arrive. The Porter jokes about drink’s effects. Macduff asks if the king is stirring; the porter claims they have been carousing. Macbeth later enters and greets Macduff and Lennox, who report that the night has been “unruly” with strange omens: storms, screaming birds, and a “feverous” earth. They discuss the murder, with Macduff horrified at the “sacrilegious” killing of the Lord’s anointed, and Lennox noting that the king has fled the castle. A frantic alarm is raised: “Murder and treason! Banquo and Donalbain! Malcolm! awake!” Macduff, Lady Macbeth, Banquo, Macbeth, Lennox, Ross, Malcolm, and Donalbain all appear in rapid succession, each reacting to the discovery of Duncan’s death and the ensuing chaos. Macbeth delivers a bleak speech about mortality and the futility of life, while Malcolm and Donalbain decide to flee Scotland—Malcolm to England, Donalbain to Ireland—believing that staying would put them in danger.

In Scene IV, Ross meets an Old Man outside the castle. The Old Man recounts dreadful omens: a falcon killed by an owl, Duncan’s horses behaving wildly, and other unnatural events. Ross and the Old Man discuss how these signs mirror the recent murder. Macduff enters, confirming that Macbeth’s murderers have fled and that Malcolm and Donalbain have escaped, making them suspects. Ross speculates that Macbeth will soon be crowned king, and Macduff mentions that Duncan’s body was taken to the sacred “Colmekill.” The scene ends with Ross and the Old Man parting, each wishing divine protection.