Alcmene belonged to the royal family of the Argolid. In Apollodorus's genealogy her father was Electryon, king of Mycenae, a son of Perseus and Andromeda. Her mother was Anaxo, daughter of Alcaeus. The closely interwoven family also included Amphitryon, whom she was to marry. The Shield of Heracles, traditionally attributed to Hesiod, praises her beauty, wisdom and devotion to her husband. Within the heroic tradition, she is both the admired wife of Amphitryon and the woman whose household becomes the focus of Zeus's desire and Hera's hostility.

Alcmene's marriage was bound to a family disaster. Her brothers had died in a conflict with the sons of Pterelaus and the Taphians or Teleboans. Before Electryon could carry out his intended campaign of vengeance, Amphitryon accidentally killed him: a club thrown at a straying cow rebounded and struck the king. Electryon's brother Sthenelus expelled Amphitryon, and Alcmene went with him to Thebes, where King Creon purified him of the killing. Alcmene insisted that her brothers must be avenged before she would share Amphitryon's bed. He therefore sought allies and sailed against the island kingdom of Pterelaus, leaving her in Thebes.

While Amphitryon was away, Zeus came to Alcmene disguised as her husband. Apollodorus says that the god made one night three times its usual length and described the victories over the Teleboans as though he had won them himself. Alcmene believed that Amphitryon had returned. The deception matters: she did not knowingly choose another partner. When the real Amphitryon came home and found that his arrival seemed to surprise her less than he expected, she explained that he had already visited her. The seer Tiresias revealed what Zeus had done. Alcmene conceived Heracles by Zeus and Iphicles by Amphitryon, twins who would share a household but not the same father.

Hera turned the approaching birth into a contest over power. Zeus had declared that a descendant of Perseus born that day would rule over his neighbours. Hera secured his oath and brought forward the birth of Eurystheus, the son of Sthenelus, while delaying Alcmene's delivery. Eurystheus was born prematurely, at seven months' gestation, and the promised supremacy passed to him. In Ovid's Roman telling, Alcmene suffers for seven days and nights while Lucina, the childbirth goddess corresponding to Eileithyia, sits outside with crossed limbs and joined fingers, maintaining the obstruction. Galanthis, Alcmene's attendant, announces that the birth has already happened. Startled, Lucina releases her posture, and the spell breaks. It is Lucina who then turns Galanthis into a weasel in this version. Greek traditions preserve other helpers and other ways in which the obstruction was broken.

Hera's hostility continued after the birth. In the familiar story, two serpents were sent against the infants, and Heracles strangled them while Iphicles cried out. Alcmene and Amphitryon raised the boys in Thebes. Apollodorus places Amphitryon's death in the war against the Minyans, in which the young Heracles also fought. Alcmene later married Rhadamanthys and lived with him at Ocaleae in Boeotia. Her life thus continued beyond the circumstances of Heracles' conception: she experienced exile, motherhood, widowhood and another marriage, while the different traditions carried her family across the Greek landscape.

The last great act of vengeance attached to Alcmene concerns Eurystheus. After Heracles' death, the king pursued his children, but the conflict ended in his defeat. Apollodorus says that Hyllus killed him and brought his severed head to Alcmene, who pierced its eyes with weaving pins. Her anger was directed at the persecutor of both her son and his descendants. Traditions of her own death differ. Pausanias reports a tomb at Megara, where the local story said that she died while travelling from Argos to Thebes; a Delphic oracle settled the family's dispute over where to bury her. Antoninus Liberalis tells a different ending: Hermes replaced her body with a stone and took her to the Isles of the Blessed to be with Rhadamanthys. Pausanias also records an altar to Alcmene at the Cynosarges in Athens, alongside the honours paid there to Heracles and his household.