The aim of this work was to evaluate the performance of a biotrickling filter (BTF) packed with polyurethane foam and pall rings for the enrichment of microorganisms mediating anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) by activity tests and microbial community analysis. A BTF was inoculated with microorganisms from a known AOM active deep sea sediment collected at a depth of 528 m below the sea level (Alpha Mound, Gulf of Cadiz). The microbial community analysis was performed by catalyzed reporter deposition - fluorescence in situ hybridization (CARD-FISH) and 16S rRNA sequence analysis. The AOM occurrence and rates in the BTF were assessed by performing batch activity assays using ¹³C-labelled methane (¹³CH₄). After an estimated start-up time of ∼20 days, AOM rates of ∼0.3 mmol l⁻¹ day⁻¹ were observed in the BTF, values almost 20 times higher than previously reported in a polyurethane foam packed BTF. The microbial community consisted mainly of anaerobic methanotrophs (ANME-2, 22% of the total number of cells) and sulfate reducing bacteria (SRB, 47% of the total number of cells). This study showed that the BTF is a suitable reactor configuration for the enrichment of microbial communities involved in AOM coupled to SR at ambient pressure and temperature with a relatively short start-up time.