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Molecular procedures were used to identify Thiothrix spp. in biofilms from sulphide-rich waters in two distinct ecosystems.Biofilm samples were obtained from two groundwater-fed systems in central and northern Florida, including an artesian spring and municipal water tank. The 16S rDNA in each sample was directly amplified by polymerase chain reaction.Clonal libraries of biofilm 16S rDNA from each site contained rDNA sequences that were 99–99·5% similar to Thiothrix unzii.This is the first report of T. unzii in a natural system. Biofilm formation by Thiothrix spp. can cause fouling in groundwater processing equipment, including municipal water-processing facilities, agricultural irrigation systems and spring water bottling plant filters. Biofouling can have severe economic and human health impacts as it will influence flow rates and related water treatments. Characterization of specific fouling bacteria and their molecular ecology is essential for their regulation.