Juan Ponce de Leon claimed Florida for Spain in 1513 after landing on the northeast coast. He returned eight years later to found a colony, but was unsuccessful and was killed. Panifilo de Narvaez anchored in Tampa Bay in 1528 and traveled inland. In 1539, Hernando de Soto landed near Tampa Bay and explored that area as well as northern Florida. Tristan de Luna attempted to establish a permanent colony on Pensacola Bay in 1559 with 1,600 men and women, but abandoned the effort after two years.

French Huguenot leader Gaspard de Coligny commissioned Jean Ribaut to found a colony in the territory in 1562. Ribaut's aide, Rene de Laudonniere, built Fort Caroline near present-day Jacksonville. Philip II of Spain sent a military expedition, led by Pedro Menendez de Aviles, to destroy the French settlement. Arriving in 1565, Menendez established St. Augustine and massacred the French. He captured Fort Caroline and founded another settlement there.