Bacterial concentration and diversity was assessed in a moderately acidic (pH 5.1) anaerobic groundwater contaminated by chlorosolvent-containing DNAPL at a Superfund site located near Baton Rouge, Louisiana. Groundwater analysis revealed a total aqueous-phase chlorosolvent concentration exceeding 1000 mg L−1, including chloroethanes, vinyl chloride, 1,2-dichloropropane, and hexachloro-1,3-butadiene as the primary contaminants. Direct counting of stained cells revealed more than 3 × 107 cells mL−1 in the groundwater, with 58% intact and potentially viable. Universal and ‘Dehalococcoides’-specific 16S rRNA gene libraries were created and analyzed. Universal clones were grouped into 18 operational taxonomic units (OTUs), which were dominated by low-G+C Gram-positive bacteria (62%) and included several as yet uncultured or undescribed organisms. Several unique 16S rRNA gene sequences closely related to Dehalococcoides ethenogenes were detected. Anaerobically grown isolates (168 in total) were also sequenced. These were phylogenetically grouped into 18 OTUs, of which only three were represented in the clone library. Phylogenetic analysis of isolates and the clone sequences revealed close relationships with dechlorinators, fermenters, and hydrogen producers. Despite acidic conditions and saturation or near-saturation chlorosolvent concentrations, the data presented here demonstrate that large numbers of novel bacteria are present in groundwater within the DNAPL source zone, and the population appears to contain bacterial components necessary to carry out reductive dechlorination.