Born into the landed gentry, he ran away to sea at age 14. In 1549 he was commissioned by the Holy Roman emperor Charles V (Charles I of Spain) to drive pirates from the coasts of Spain. Five years later he was appointed captain of the Indies fleet. An impatient man who acted with dispatch and reluctantly concerned himself with administrative details, he made numerous enemies, who brought about his imprisonment in 1563. He was freed two years later after he had regained royal favour.

Because King Philip II was disturbed by the potential threat to Spain's possessions from a settlement of French Huguenots on a strategic part of the Florida coast, he sent Menéndez de Avilés to Florida to establish a colony there and deal with the French. The expedition sailed in July 1565 with 11 ships and about 2,000 men. On August 28 he entered and named the bay of St. Augustine and built a fort there. On September 20 he took the nearby French colony of Fort Caroline and massacred the entire population, hanging the bodies on trees with the inscription “Not as Frenchmen, but as heretics.” Menéndez de Avilés then explored the Atlantic coast and established a string of forts as far north as the island of St. Helena (off present-day South Carolina). He was recalled to Spain in 1567 and later helped organize a squadron of ships against the English. He died while engaged in this task.