A Huguenot exploratory group led by French naval officer Jean Ribault had landed at the site on the River of May (now the St. Johns River) in February 1562. The exploration had then moved north to Port Royal Sound, established Charlesfort on Parris Island and stayed for a time. Ribault returned to Europe to arrange supplies for the new colony, but was arrested in England due to complications arising from the French Wars of Religion.
Without supplies or leadership, and beset by hostility from the native Timucua, most of the colonists followed René Goulaine de Laudonnière south, where they founded Fort Caroline (or Fort de la Caroline) atop St. Johns Bluff on June 22, 1564. The fort was named for the reigning French king, Charles IX.
In August 1565, Ribault returned to Ft. Caroline to take command of the settlement. Upon learning of the Spanish colony of St. Augustine just 35 mi (60 km) to the south, Ribault set out with several ships carrying 200 sailors and 400 soldiers to dislodge the Spanish, but he was surprised at sea by a violent storm lasting several days. Don Pedro Menéndez de Avilés, the governor of Florida for Spain, took advantage of this. Marching his troops overland, he surprised, at dawn on September 20, the Fort Caroline garrison which then numbered about 200 to 250 people. The Spaniards attacked and killed most of the defenders. The only survivors were about 50 women and children who were taken prisoner and 26 defenders, including de Laudonnière, who managed to escape. As for the men of Ribault's fleet, several had drowned; the Spanish picked up about 350 survivors, (including Ribault), south on the coast where their ships had been wrecked, only to put them to the sword, sparing about 20 (not including Ribault). This place is known today by a fort built much later, Fort Matanzas (Fort Massacre). This massacre put an end to France's attempts at colonization in Florida.
The Spanish continued to occupy the fort. In April 1568, Dominique de Gourgues led a French force which attacked and burned the fort. The Spanish rebuilt it, only to permanently abandon it the next year. The exact location of the settlement is not known.
In early September 1565, Ribault sails with his small squadron and detachment of soldiers for St. Augustine. Despite a storm which scatters his ships he sights the Spanish fort on September 13 and offloads the majority of his men out of sight on the north shore. By the time he has landed the bulk of his force the missing ships have rejoined his squadron. Directing his ships to bombard the Spanish to keep their attention he hits the garrison where they least expect it, the landward side of the fort. The battle is furious with both Ribault and the Spanish commander, Don Pedro Menéndez, slain. The Spanish yield the fort and with it all claim to Florida.
In 1720 Fort Caroline is still an important garrison for the French colony of Florida, though the settlement has long outgrown the confines of the original earth and pallisade works that protected it.