Born into a noble family, Ponce de León was a page in the royal court of Aragon and later fought in a campaign against the Moors in Granada. It is possible that he began his career of exploration in 1493 as part of Christopher Columbus' second expedition to the New World. In 1502 he was in the West Indies as a captain serving under Nicolás de Ovando (Ovando, Nicolás de), governor of Hispaniola. As a reward for suppressing an Indian mutiny, Ponce de León was named by Ovando to be the provincial governor of the eastern part of Hispaniola. Hearing persistent reports of gold to be found on Puerto Rico, Ponce de León in 1508–09 explored and settled that island, founding the colony's oldest settlement, Caparra, near what is now San Juan. He then returned to Hispaniola and was named governor of Puerto Rico but was soon displaced from the governorship through the political maneuvering of rivals.

The Spanish crown encouraged Ponce de León to continue searching for new lands. He learned from Indians of an island called Bimini (in the Bahamas) on which there was a miraculous spring or fountain that could rejuvenate those who drank from it (the fountain of youth). In search of this fountain, he led a privately outfitted expedition from Puerto Rico in March 1513 and in April of that year landed on the coast of Florida near the site of modern St. Augustine. At the time he did not realize that he was on the mainland of North America and instead supposed he had landed on an island. He named the region Florida because it was discovered at Easter time (Spanish: Pascua Florida) and because it abounded in lush, florid vegetation. He coasted southward, sailing through the Florida Keys and ending his search near Charlotte Harbor on Florida's west coast. He then returned to Puerto Rico and thence to Spain, where he secured the title in 1514 of military governor of Bimini and Florida with permission to colonize those regions.

In 1521 Ponce de León sailed again for Florida with two ships and 200 men, landing near Charlotte Harbor. On this occasion he was wounded by an arrow during an Indian attack, and he died after being returned to Cuba. Puerto Rico's third largest city, Ponce, is named in his honour.