Polycyclic aromatic hydrocarbons (PAH) are widespread and persistent environmental contaminants, especially in oxygen-free environments. The occurrence of anaerobic PAH-degrading bacteria and their underlying metabolic pathways are rarely known. In this study, PAH degraders were enriched in laboratory microcosms under sulfate-reducing conditions using groundwater and sediment samples from four PAH-contaminated aquifers. Five enrichment cultures were obtained showing sulfate-dependent naphthalene degradation. Mineralization of naphthalene was demonstrated by the formation of sulfide concomitant with the depletion of naphthalene and the development of ¹³C-labeled CO₂ from [¹³C₆]-naphthalene. 16S rRNA gene and metaproteome analyses revealed that organisms related to Desulfobacterium str. N47 were the main naphthalene degraders in four enrichment cultures. Protein sequences highly similar to enzymes of the naphthalene degradation pathway of N47 were identified, suggesting that naphthalene was activated by a carboxylase, and that the central metabolite 2-naphthoyl-CoA was further reduced by two reductases. The data indicate an importance of members of the family Desulfobacteraceae for naphthalene degradation under sulfate-reducing conditions in freshwater environments.