A giant 800-m-diameter pockmark named REGAB was discovered on the Gabon continental margin actively emitting methane at a water depth of 3200 m. The microbial diversity in sediments from four different assemblages of chemosynthetic organisms, Mytilidae, Vesicomyidae, Siboglinidae and a bacterial mat, was investigated using comparative 16S rRNA gene sequence analysis. Aggregates of anaerobic methanotrophic archaea (ANME-2) and bacteria of the Desulfosarcina/Desulfococcus cluster were found in all four chemosynthetic habitats. Fluorescence in situ hybridization targeting the ANME-2/Desulfosarcina/Desulfococcus aggregates showed their presence few centimeters (3-5 cm) below the surface of sediment. 16S rRNA gene sequences from all known marine ANME groups were detected in the pockmark sediments, as well as from both known bacterial partners. The archaeal diversity was limited to the ANME cluster for all investigated samples. The bacterial diversity included members of the Proteobacteria, Bacilliales, Cytophaga/Flavobacteria, Verrucomicrobia, JS1 and Actinobacteria clusters. Bacterial 16S rRNA gene sequences related to those of known sulphide-oxidizing symbionts were recovered from tissues of several invertebrates including vesicomyid clams and siboglinid tubeworms of REGAB.