Our case study analyzed the proximity of previously mapped fractures in the aquifer matrix to 93 Florida panther (Puma concolor coryi) dens mapped from 2007-2016 in south Florida. Dens occurred in five counties (Collier = 77, Dade = 1, Hendry = 9, Lee = 5, and Monroe = 1) and three sub-basins of the Greater Everglades Basin (Big Cypress Swamp = 83, Caloosahatchee = 3, and Everglades = 7). Fractured aquifers occur worldwide, but are not the focus of habitat suitability studies, despite evidence that fractures influence plant species composition and density. Habitat alterations can occur many kilometers from the surface footprint of groundwater alterations in the regional Floridan aquifer system via preferential flow through fractures. Increased natural discharge from and recharge to the aquifer occur at fracture intersections. Greater induced recharge and habitat changes also may occur at fracture intersections. All dens were within 5 km of a previously mapped fracture; 36% and 74% were within 1 km and 2 km, respectively, of those fractures; and 47%, 74%, and 90% of dens were within 2 km, 3.25 km and 5 km, respectively, from the nearest fracture intersection. Results suggest fractures influence the suitability and/or availability of habitat for panther dens, selection of den sites, and availability as well as abundance of high quality prey items essential for the nutritional demands of successfully rearing panther kittens in the wild. We recommend more detailed investigations of: a) vegetation characteristics near dens, b) groundwater alterations and cumulative impacts of those alterations associated with fractures in panther habitat (e.g., altered plant species composition and density), and c) influence of aquifer fractures in all habitats underlain by fractures.