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Thiothrix spp., sulfide‐oxidizing mixotrophic bacteria, were sampled from visible colonies in the Floridan aquifer in several underwater caves, sinkholes, and springs below the water table in North Florida. Bacteria samples were collected by cave divers certified by the National Speleological Society/Cave Diving Section. Sites sampled were ecological niches in the aquifer where visible colonies had a white slimy or filamentous appearance indicative of Thiothrix spp. Sterile sampling methods were adapted to the underwater cave setting. Bulk water samples for media preparation were collected by divers from bacteria sampling sites. Bacteria were isolated and cultured in growth media prepared with cave or spring water. Thiothrix spp. were identified by microbiological and immunological methods. Monoclonal antibodies specific for Thiothrix spp. were utilized in fluorescent antibody assays and enzyme‐linked im‐munosorbent assays (ELISA). Thiothrix was found in six of eight underwater caves sampled. Three of the...