Mark Antony – one of the triumvirs of the Roman Republic, along with Octavius and Lepidus – has neglected his soldierly duties after being beguiled by Egypt's Queen, Cleopatra. He ignores Rome's domestic problems, including the fact that his third wife Fulvia rebelled against Octavius and then died.
Octavius calls Antony back to Rome from Alexandria to help him fight against Sextus Pompey, Menecrates, and Menas, three notorious pirates of the Mediterranean. At Alexandria, Cleopatra begs Antony not to go, and though he repeatedly affirms his deep passionate love for her, he eventually leaves.
The triumvirs meet in Rome, where Antony and Octavius put to rest, for now, their disagreements. Octavius' general, Agrippa, suggests that Antony should marry Octavius's sister, Octavia, in order to cement the friendly bond between the two men. Antony accepts. Antony's lieutenant Enobarbus, though, knows that Octavia can never satisfy him after Cleopatra. In a famous passage, he describes Cleopatra's charms: "Age cannot wither her, nor custom stale / Her infinite variety: other women cloy / The appetites they feed, but she makes hungry / Where most she satisfies."
A soothsayer warns Antony that he is sure to lose if he ever tries to fight Octavius.
In Egypt, Cleopatra learns of Antony's marriage to Octavia and takes furious revenge upon the messenger that brings her the news. She grows content only when her courtiers assure her that Octavia is homely: short, low-browed, round-faced and with bad hair.
Before battle, the triumvirs parley with Sextus Pompey, and offer him a truce. He can retain Sicily and Sardinia, but he must help them "rid the sea of pirates" and send them tributes. After some hesitation Sextus agrees. They engage in a drunken celebration on Sextus' galley, though the austere Octavius leaves early and sober from the party. Menas suggests to Sextus that he kill the three triumvirs and make himself ruler of the Roman Republic, but he refuses, finding it dishonourable. After Antony departs Rome for Athens, Octavius and Lepidus break their truce with Sextus and war against him. This is unapproved by Antony, and he is furious.
Antony returns to Alexandria and crowns Cleopatra and himself as rulers of Egypt and the eastern third of the Roman Republic (which was Antony's share as one of the triumvirs). He accuses Octavius of not giving him his fair share of Sextus' lands, and is angry that Lepidus, whom Octavius has imprisoned, is out of the triumvirate. Octavius agrees to the former demand, but otherwise is very displeased with what Antony has done.
Antony prepares to battle Octavius. Enobarbus urges Antony to fight on land, where he has the advantage, instead of by sea, where the navy of Octavius is lighter, more mobile and better manned. Antony refuses, since Octavius has dared him to fight at sea. Cleopatra pledges her fleet to aid Antony. However, during the Battle of Actium off the western coast of Greece, Cleopatra flees with her sixty ships, and Antony follows her, leaving his forces to ruin. Ashamed of what he has done for the love of Cleopatra, Antony reproaches her for making him a coward, but also sets this true and deep love above all else, saying "Give me a kiss; even this repays me."
Octavius sends a messenger to ask Cleopatra to give up Antony and come over to his side. She hesitates, and flirts with the messenger, when Antony walks in and angrily denounces her behavior. He sends the messenger to be whipped. Eventually, he forgives Cleopatra and pledges to fight another battle for her, this time on land.
On the eve of the battle, Antony's soldiers hear strange portents, which they interpret as the god Hercules abandoning his protection of Antony. Furthermore, Enobarbus, Antony's long-serving lieutenant, deserts him and goes over to Octavius' side. Rather than confiscating Enobarbus' goods, which Enobarbus did not take with him when he fled, Antony orders them to be sent to Enobarbus. Enobarbus is so overwhelmed by Antony's generosity, and so ashamed of his own disloyalty, that he dies from a broken heart.
Antony loses the battle as his troops desert en masse and he denounces Cleopatra: "This foul Egyptian hath betrayed me." He resolves to kill her for the treachery. Cleopatra decides that the only way to win back Antony's love is to send him word that she killed herself, dying with his name on her lips. She locks herself in her monument, and awaits Antony's return.
Her plan backfires: rather than rushing back in remorse to see the "dead" Cleopatra, Antony decides that his own life is no longer worth living. He begs one of his aides, Eros, to run him through with a sword, but Eros cannot bear to do it and kills himself. Antony admires Eros' courage and attempts to do the same, but only succeeds in wounding himself. In great pain, he learns that Cleopatra is indeed alive. He is hoisted up to her in her monument and dies in her arms.
Octavius goes to Cleopatra trying to persuade her to surrender. She angrily refuses since she can imagine nothing worse than being led in chains through the streets of Rome, proclaimed a villain for the ages. She imagines that "the quick comedians / Extemporally will stage us, and present / Our Alexandrian revels: Antony / Shall be brought drunken forth, and I shall see / Some squeaking Cleopatra boy my greatness / I' th' posture of a whore." This speech is full of dramatic irony, because in Shakespeare's time Cleopatra really was played by a "squeaking boy" and Shakespeare's play does depict Antony's drunken revels.
Cleopatra is betrayed and taken into custody by the Romans. She gives Octavius what she claims is a complete account of her wealth but is betrayed by her treasurer, who claims she is holding treasure back. Octavius reassures her that he is not interested in her wealth, but Dolabella warns her that he intends to parade her at his triumph.
Cleopatra kills herself using the venomous bite of an asp, imagining how she will meet Antony again in the afterlife. Her serving maids Iras and Charmian also die, Iras from heartbreak and Charmian from another asp. Octavius discovers the dead bodies and experiences conflicting emotions. Antony's and Cleopatra's deaths leave him free to become the first Roman Emperor, but he also feels some sympathy for them. He orders a public military funeral.