The U.S. District Court for the Northern District of Florida serves the residents of twenty-three counties. These are Alachua, Bay, Calhoun, Dixie, Escambia, Franklin, Gadsden, Gilchrist, Gulf, Holmes, Jackson, Jefferson, Lafayette, Leon, Levy, Liberty, Madison, Okaloosa, Santa Rosa, Taylor, Wakulla, Walton, and Washington counties.

The four courthouses in which it hears cases are Gainesville, Panama City, Pensacola, and Tallahassee.