According to the Bibliotheca, Alcmene was born to Electryon, son of Perseus and Andromeda and king of Tiryns and Mycenae. Her mother was Anaxo, daughter of Alcaeus and Astydamia. Other accounts name her mother as Lysidice (daughter of Pelops and Hippodameia) or Eurydice (daughter of Pelops), while the poet Asius made her the daughter of Amphiaraus and Eriphyle.

Hesiod describes Alcmene as remarkably beautiful and exceptionally wise. Her appearance rivaled Aphrodite's, and she honored her husband as no woman before her had.

Alcmene accompanied Amphitryon to Thebes, where he was purified by Creon for accidentally killing her father, Electryon. She refused to marry Amphitryon until he had avenged the deaths of her brothers.

While Amphitryon was away fighting the Taphians and Teleboans, Zeus desired Alcmene and extended a single night into three days by commanding Helios not to rise. Disguised as Amphitryon, Zeus persuaded Alcmene that he was her husband and lay with her, conceiving Heracles as he recounted his military victories. When Amphitryon returned, Alcmene told him of the previous night's encounter, and Amphitryon learned from the seer Tiresias what Zeus had done.

In Homer's Iliad, when Alcmene was about to give birth, Zeus announced to the gods that a child of his own would be born and rule over all those around him. Hera made him swear an oath, then descended to Argos and caused the wife of Sthenelus to give birth to Eurystheus after only seven months, while at the same time preventing Alcmene from delivering Heracles. So it was Eurystheus, not Heracles, who came to rule.

According to Ovid's Metamorphoses, Alcmene endured seven days and nights of labor while bearing so large a child. She called upon Lucina, goddess of childbirth, but Juno had ordered Lucina to obstruct the delivery. With clasped hands and crossed legs, Lucina chanted spells that blocked the birth, and Alcmene nearly died. Her maid Galanthis, noticing the goddess's posture, deduced Juno's involvement and announced that Alcmene had already given birth safely. Lucina sprang up in surprise, unclenching her hands and breaking the spell, so that Heracles was born at once. As punishment, Galanthis was transformed into a weasel but continued to live with Alcmene.

Pausanias relates that Hera sent witches (so called by the Thebans) to prevent the delivery. They succeeded until Historis, daughter of Tiresias, used a trick like that of Galanthis, announcing the child's safe birth and deceiving the witches into leaving, which allowed Alcmene to give birth.

By contrast, Plautus's comedy Amphitryon offers a gentler version: Alcmene calls upon Jupiter, who works a miracle so that, after a crash of thunder and light, the child arrives quickly, painlessly, and without assistance.

After Amphitryon's death, Alcmene married Rhadamanthys, a son of Zeus, and lived with him in exile at Ocaleae in Boeotia. Following Heracles' apotheosis, Hyllus pursued and killed Eurystheus and presented his head to Alcmene, who gouged out its eyes with weaving pins. In the Metamorphoses, an aged Alcmene recounts the story of Heracles' birth to Iole.

Two accounts describe her death. According to the Megarians, Alcmene died while walking from Argos to Thebes, at Megara. The Heracleidae disagreed over whether to return her body to Argos or bury it at Thebes alongside Amphitryon and Heracles' children by Megara; the Delphic oracle advised burial at Megara. The Theban account claims that on her death she was transformed from human form into stone.

Pausanias noted that an altar to Alcmene stood at the Cynosarges in Athens alongside altars to Heracles, Hebe, and Iolaus. Her tomb is located near the Olympieum at Megara.