Rouffi provided Rojas with a complete account of Ribault's endeavor. With Rouffi acting as guide, Rojas went to Charlesfort where he found both the earthwork and a standing, empty building. The Spaniards burned the building, but Rojas did not certify in his report that he filled the moat, although his orders were to raze the fort "so completely that no trace of it shall remain." After he had burned the building, Rojas sailed to a nearby island where he found the stone marker. He and his men loaded the stone column onto their boat, and shortly thereafter the Spaniards sailed for Cuba. Rojas departed from Port Royal on June 15, sailed toward Cuba by way of the Lucayas (because winds would not allow him to sail directly south along the Florida coast, and arrived in Havana on July 9, 1564.
As Rojas sailed for home, he narrowly missed an encounter with a second French expedition to Florida. René de Laudonnière, with three ships and more than three hundred men, had sailed from Le Harve April 22, 1564. Two months later, on June 22, his vessels entered the River May (St. Johns) near present day Jacksonville, Florida. After reestablishing relations with local Timucua Indians, Laudonnière began construction of Fort Caroline on the south bank of the River May. Over the next several months, the Frenchmen in Fort Caroline suffered hunger and hardship. Laudonnière lost one of his vessels to mutineers, but he ultimately he was able to quell the rebellion and execute the ringleaders. In January, 1565, a ship was dispatched to Port Royal to rescue Rouffi. Laudonnière had no way of knowing that he had been picked up by Spaniards six months earlier. The Frenchmen in Fort Caroline expected Jean Ribault to arrive in spring, 1565, with supplies and reinforcements, but he did not show up. By June, Laudonnière had given up on any chance of resupply, and he began preparations to abandon his Florida colony. On August 28, just as Laudonnière was ready to sail for home, Ribault arrived with a relief fleet of five vessels and about 800 men. Ribault assumed command, and began immediately to make repairs to the partially dismantled fort.
Ribault's efforts were in vain, however. Early in September, Spaniard Pedro Menéndez de Avilés arrived on the coast of Florida with a large force consisting of more than one thousand men on ten ships. Menéndez had been dispatched by Philip II of Spain to drive the French from Florida, and he wasted little time in carrying out his orders. After an initial skirmish with Ribault's ships, Menéndez mounted a land assault against Fort Caroline on the morning of September 20, 1565. In a raging storm, Menéndez and his men routed the French garrison and captured their fort. One hundred thirty-two Frenchmen were killed in the assault, and 45 escaped; Menéndez ordered that women and children should not be killed, and fifty of them were captured. After strengthening the ramparts of the captured fort, he renamed it Fort San Mateo and set off to round up the Frenchmen who had escaped during the attack.
At Matanzas Inlet south of St. Augustine, Menéndez found a group of Frenchmen whose ship had wrecked on the coastline. When these men surrendered to Menéndez, he spared the lives of 17, but more than one hundred others were killed. In mid-October, the Spaniards learned that another group of French survivors from Fort Caroline had wrecked near Matanzas. Menéndez convinced these men, including Jean Ribault, to surrender: they, like the others, were mercilessly killed.
With the destruction of the French settlement completed, Pedro Menéndez turned his attention to establishing his own stronghold in Florida. He strengthened the defenses at St. Augustine which he had established as a base from which to mount his assault on Fort Caroline, and he reinforced Fort San Mateo. Then, in the spring of 1566, he established the town of Santa Elena on Parris Island in order to prevent future French incursions in that harbor. Menéndez built the first Santa Elena fort, San Salvador, to house the men he left to defend his new settlement.
In July, 1566, Captain Juan Pardo arrived at Santa Elena with 250 men under his command. Denied entrance into Fort San Salvador by its commander, Esteban de las Alas, Pardo constructed his own fort, the first of two forts named San Felipe; this fort will hereafter be called San Felipe (I). Pardo was soon dispatched into the interior on a mission of exploration, and Fort San Felipe (I) appears from extant accounts to have become the primary fort at Santa Elena. Pardo's fort was occupied until 1570, when the blockhouse within its walls accidentally burned. A new fort, the second Fort San Felipe (II), was built elsewhere in Santa Elena, and Pardo's Fort San Felipe was never reoccupied. Fort San Felipe (II) was occupied until 1576, when an Indian attack forced both the fort's garrison and the residents of the town of Santa Elena to flee to St. Augustine. When Santa Elena was reoccupied in 1577, a new fort, San Marcos, was built. The Spanish occupation at Santa Elena continued until 1587, when the town was abandoned and never reoccupied.