Lysistrata is an intelligent, unmarried Athenian woman who devises a radical plan to halt the ongoing Peloponnesian War. Unlike her peers, she remains untroubled by the need for physical intimacy, focusing instead on civic duty and peace. She reads an oracular text to keep the women motivated and directly subdues the local authorities using both logic and household items. She acts with courage and strategic thinking, establishing her as a distinct figure from traditional Greek housewives.

Lampito is a physically fit Spartan woman who acts as Lysistrata's counterpart across enemy lines. She possesses a pragmatic attitude and political savvy, making her the first to recognize the logic in the sex embargo. Her physical strength is a source of marvel and mild flirtation for the Athenian women, and she takes responsibility for organizing the strike in her home region.

The Chorus consists of twenty-four older Athenians divided evenly by gender. The old men are military veterans who currently serve as jurors, carrying torches to reclaim the Acropolis by force. The old women operate as upstanding civil servants who intercept the men with water, prepared to extinguish their physical threats. They provide a constant commentary on the unfolding events, trading insults and historical analogies throughout the conflict.

The Councilor is a member of the emergency political council who represents the stubborn, war-mongering leadership of Athens. He immediately clashes with the occupying women and attempts to arrest them. He is dismissive of female intelligence, blames lenient husbands for the uprising, and prioritizes military funding over diplomatic solutions.

Calonice is an Athenian housewife who serves as a sounding board for Lysistrata's initial plotting. She embodies typical social expectations for women in the city, frequently making suggestive jokes and expressing reluctance to abandon her luxurious attire for the cause.

Myrrhine is a young Athenian wife who joins the political embargo at the Acropolis. She struggles with the severe demands of the strike but follows instructions to mercifully tease her frustrated husband without breaking her solemn oath. When he arrives to see her, she pretends to be highly particular about arranging a cot for their intimacy, drawing out his anticipation.

Cinesias is Myrrhine's hapless husband who comes to the Acropolis begging for his wife to return home. He wears a comically oversized apparatus demonstrating his physical agony and attempts to use their hungry infant child as leverage to break her resolve.

The Spartan Herald acts as a messenger who arrives in Athens seeking diplomatic talks. He suffers from the same severe physical desperation as the local Athenian men, proving that the embargo functions successfully across enemy lines and forcing the men into negotiations.

The Goddess of Deals is a personification of peace represented by an unclad woman. She serves as a highly distracting visual aid during the treaty discussions, easily capturing the attention of the desperate diplomats from both sides.

Cleisthenes is an Athenian man known for his effeminate behaviors. Though he does not directly participate in the action, he serves as a frequent punchline for the older men regarding the desperate measures they might resort to if the women's strike continues indefinitely.