This study was performed to enrich anaerobic methane-oxidizing archaea (ANME) present in sediment from the Ginsburg Mud Volcano (Gulf of Cadiz) in a polyurethane foam packed biotrickling filter (BTF). The BTF was operated at 20 (+/- 2) degrees C, ambient pressure with continuous supply of methane for 248 days. Sulfate reduction with simultaneous sulfide production (accumulating similar to 7 mM) after 200 days of BTF operation evidenced anaerobic oxidation of methane (AOM) coupled to sulfate reduction. High-throughput sequence analysis of 16S rRNA genes showed that after 248 days of BTF operation, the ANME clades enriched to more than 50% of the archaeal sequences, including ANME-1b (40.3%) and ANME-2 (10.0%). Enrichment of the AOM community was beneficial to Desulfobacteraceae, which increased from 0.2% to 1.8%. Both the inoculum and the BTF enrichment contained large populations of anaerobic sulfur oxidizing bacteria, suggesting extensive sulfur cycling in the BTF.