Act II opens with Banquo and his son, Fleance, making their way to bed in Macbeth's castle. Macbeth emerges from the darkness, and speaks to Banquo. Banquo tells Macbeth that he recently had a dream about the witches and the prophecies, mentioning, in particular, that one of Macbeth's prophecies has come true in some regard. Macbeth feigns disinterests in the topic, and the two agree to discuss the witches at a later time. However, Fleance's presence is a reminder to the audience that, though Macbeth might be king, it will not be a position he keeps if the witches' prophecies are to be believed.

After Fleance and Banquo leave, Macbeth is alone on stage. In one of the most famous soliloquys in the play, Macbeth sees a hallucination of the dagger, seeming to point the way toward Duncan's murder. It also seems to him that the handle is pointed toward his hand, as if it beckons him to clutch it and complete Duncan's murder. However, he concludes that it must just be only hallucination, a manifestation of his feelings of guilt in his plans to kill Duncan. But this is not the first hallucination that will appear in the play. At the end of this scene, Macbeth hears a ringing of a bell, which is Lady Macbeth's signal that they should commence their murder plans.

The next scene begins with Lady Macbeth. She has already drugged the guards and is waiting for Macbeth to complete the murder. She remarks that, if Duncan hadn't looked so much like her own father, she might have murdered him herself. This is the first time that Lady Macbeth shows any sign of a conscience. However, her comment also places the responsibility for the physical murder solely upon Macbeth.

Macbeth emerges from Duncan's chambers, covered in blood. Duncan's actual murder is never seen in the play, only the events leading up to it and its aftermath. Clearly shaken by what he has done, Macbeth rambles on about how he has "murdered sleep" and how disturbed he was that he could not say "amen" to the guards' prayers. He feels haunted and cursed. Lady Macbeth scolds him, enraged when she sees that he has bought the murder weapons with him. She calls him a coward and takes the daggers from him, delivering them back to the scene of the crime. She returns, also covered in blood, and remarks how easily water will wash away their crimes. Meanwhile, a knocking sound can be heard throughout the castle. At the end of this scene Macbeth states, "wake Duncan with thy knocking, I would thou couldst." Here, he essentially says that he wishes the knocking would wake Duncan up, showing that he does indeed feel regret for what he has done.

Scene 3 marks a distinct change of pace. Shakespeare often incorporated scenes of comic relief into his plays, especially his tragedies. These scenes are designed to break the dramatic tension with a short, humorous scene. In the case of Macbeth, the comic relief comes in the form of the doorkeeper to the castle, the porter. He is clearly hungover from the revelry of the night before, and he pretends that he is the gatekeeper of hell. He does eventually open the door, letting in Macduff and Lennox, two of Duncan's thanes. The porter continues to ramble humorously on about the effects of alcohol to them.

Macbeth enters the scene, and Macduff asks if Duncan is yet awake. Macduff goes off to see the king, and Lennox remarks on the fierce storm that raged the night before. Macduff comes running back to report that Duncan has been murdered. Immediately, all of the thanes rush to Duncan's chambers, of course finding him dead. Lady Macbeth arrives at the scene, feigning her own disbelief, even going so far as to pretend to faint. The guards, covered in blood and clueless about the previous night's events, are immediately blamed. Macbeth, however, kills the guards, claiming his rage made him do so. Duncan's sons, Malcolm and Donalbain, arrive some time later. They discuss the murderer, wondering if their own lives are in danger. They agree that, for their own safety, they should flee the scene. Malcolm decides to flee to England, while Donalbain decides to flee to Ireland.

Act II ends with a short scene, showing Ross-one of the thanes-walking with an old man. They discuss the strange storm of the night before and the curious behavior of Duncan's horses which have, allegedly, run wild and eaten each other. Macduff emerges from the castle to tell Ross that the thanes have decided Macbeth should be the next king and that they are accompanying him to Scone to be crowned. Meanwhile, blame has been placed on Malcolm and Donalbian for the murders, since they have suspiciously disappeared from the scene. It is supposed they paid the guards to kill Duncan.

As with the first scene with the witches, the weather emerges in this act as an important element. Just as the storm in the first scene of the play suggested something was amiss with the witches, so this one suggested something was amiss in the castle. Additionally, the fact that Duncan's horses attacked each other is also symbolic. The horses, servants to Duncan, are rather like his thanes. Their fighting seems to mimic the discord that Macbeth has so recently created.

Additionally, blood is a prominent symbol in this scene. Both Macbeth and Lady Macbeth emerge from the crime covered in blood. Macbeth mentions a desire to wash the blood from his hands, and Lady Macbeth comments on how easy it is to get rid of the blood, the proof of their crimes. Blood as a symbol, especially one of guilt, will persist throughout the play.

Also in this scene, Macbeth and Lady Macbeth's characters are further developed. Lady Macbeth once again chides Macbeth for his lack of courage, questioning his manliness after he shows remorse over the murder. However, Lady Macbeth also shows for the first time that she is not without conscience. She could not kill Duncan herself because he looked like her father, someone she presumably loved. Though in the previous act she came across as a heartless woman, defying the expectations of her time, she is typical in at least one regard: love for her family.

Macbeth, at the very end of the act, also shows change. While he at first appears agitated leading up to the discovery of Duncan's murderer, he immediately springs into action after it is revealed. Thinking ahead, he kills to guards to prevent them from asserting their innocence, he helps to shift the blame to Malcolm and Donalbain, and he manages to get himself elected as king by the other thanes.