What is the most important theme in the play Macbeth?
Much of Macbeth's and Lady Macbeth's struggles in the play have to do with how they deal with the guilt of their sins. That guilt which, for both characters, turns into anxiety and fear, and back into guilt again, is brought on by their sins. So, there are a number of themes here: guilt, anxiety, and sin. But since those are all the result of ambition, one could argue that ambition is one of the most important themes in the play; maybe it is the most important.
When the witches (weird sisters) give Macbeth the first prophecies, Macbeth is skeptical until Ross informs him that he's been given the title, "Thane of Cawdor," thus confirming the witches' prophecy. Macbeth is still reluctant to take part in going after the crown, but he warms up to the idea slowly. Lady Macbeth is perhaps more ambitious than Macbeth. When Macbeth is reluctant to kill Duncan, she encourages him. But like Macbeth, she must also psyche herself into becoming less conscious of any guilt or sin she might experience. In her famous soliloquy, she asks to be "unsexed" so that she might be less feminine, less human and more inclined to remorseless action:
Come, you spirits
That tend on mortal thoughts, unsex me here,
And fill me from the crown to the toe top-full
Of direst cruelty. Make thick my blood,
Stop up th'access and passage to remorse, (I.v.38-42)
Lady Macbeth relies on her own ambition to overcome any guilt or remorse she might feel, having committed sin. Macbeth is consumed with guilt but he continues killing in order to remove any threats to his crown and thereby remove any possibilities of his sins being discovered. Here, Macbeth relies on ambition (of keeping the throne) in order to eradicate his fears (which stem from his guilt). For Macbeth, ambition is not just the guiding motivation during his murderous reign; ambition is also the tool he and Lady Macbeth use to justify their actions and they also use it to overcome the guilt that is the effect of that ambition. In the end, the guilt and/or fear never goes away.