There they learned that the rival French had already built a fort on the River of May. That fortification, they soon learned, was called Fort Caroline, defiantly honoring the reigning King of France, Charles IX. It was a visible threat to Spain's future in the New World. Moreover, the French military forces and the colonists already settled in that area, it was soon learned, were largely Huguenots.

Menéndez made a rapid sortie to the north and ascertained the position of the French interlopers, after which he returned south to the site already selected for the Spanish settlement. Plans had, in the meantime, been elaborated for the act of formal possession of the land and the installing of a legal government. On September 8, ceremoniously landing from the ship anchored in the harbor, Pedro Menéndez de Avilés went through the ritual of taking possession of La Florida in the name of the King of Spain. Sworn in as Adelantado, Captain-General and Governor of La Florida, he took solemn possession of the land.

By establishing a municipal government, Spain's claim to the land was legally confirmed. All these civil acts were solemnized with a Mass, offered by Father Francisco Lópes de Mendoza, who shortly was to become the Pastor of the new parish. A solemn Te Deum was sung. The site of the first landing was christened Nombre de Dios, and the site of the colony was named San Agustin' in honor of the Saint on whose feast day Menéndez had first viewed the harbor at which it was settled. Thus was founded the first permanent European colony in the area of the future United States.

On September 18, with a detachment of some 500 soldiers Menéndez surreptitiously set out from the new settlement on foot for the French Fort Caroline. In a surprise attack on September 20 he took the fort. The French were slaughtered mercilessly, over 130 common soldiers being slain, while many officers were held for ransom. In the hands of the Spaniards, the fort was re-christened Fort San Mateo, in honor of the Evengelist-Saint on whose feast, September 28, it finally fell into the hands of the Spaniards.⁷