Louisiana became a Spanish colony in the 1760s.Church jurisdiction was transferred to the Diocese of Santiago de Cuba and later Havana. In 1793, the vast Diocese of Louisiana and the Floridas was created; the diocese stretched from Key West Florida, below the American colonies and west of the Mississippi River into Canada. Luis Peñalver y Cárdenas, a native of Havana, was named by the King of Spain as the first bishop. He arrived in New Orleans in 1795.

Church-state relations during the Spanish period were governed by the patronato real. The king had the right to establish dioceses, to nominate bishops, and to authorize missionaries to serve in the colony. In turn, the crown purchased and erected churches and schools, paid clergy, religious, and sacristan salaries, and even enforced Church laws such as Sunday observance.

New Catholic parishes continued to be established, including St. John the Baptist Parish in Edgard (1772) and St. Bernard Parish below New Orleans (1787).

Louisianians, led by Governor Bernardo de Gálvez, joined the Anglo-American colonists in their War of Independence from England. The British were defeated in a series of encounters at Manchac, Baton Rouge, Mobile, and finally, in 1781, Pensacola.

In 1788, New Orleans experienced the first of two devastating fires. St. Louis Church was destroyed in the conflagration. A new church was completed and put in use in 1794, thanks to the generous donation of Don Andrés Almonester y Roxas.

By 1800, New Orleans was one of North America's most cosmopolitan cities. Of the 724 children and adults baptized at St. Louis Cathedral that year, fifty-two percent (52%) were slaves, thirty-one percent (31%) were whites, and seventeen percent (17%) were free persons of color. The native-born population was increased by natives of France, Spain, the Canary Islands, Cuba, Mexico, Bohemia, Italy, Canada, Scotland, Jamaica, Santo Domingo, Spanish Morocco, Puerto Rico, Martinique, Flanders and Ireland as well as the African continent.

In 1801, Bishop Peñalver was named the Archbishop of Guatemala. His departure soon left a void in ecclesiastical authority. The Louisiana Purchase two years later changed the direction of Louisiana Catholicism.

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