Bacteria that belong to the genus Desulfitobacterium are able to cleave the ether bond of lignin-borne phenyl methyl ethers via O-demethylation of the methoxy moiety. Phenyl methyl ethers occur naturally in soils and originate from lignin degradation by white rot fungi under aerobic conditions. In order to assess the possible involvement of the anaerobic desulfitobacteria in the network of lignin degradation in soils, their participation in the O-demethylation of phenyl methyl ethers in the environment was studied. In growth experiments, several Desulfitobacterium spp. were able to O-demethylate the phenyl methyl ethers 4-hydroxyanisole, syringate, vanillate and isovanillate in the presence of fumarate, nitrate, thiosulfate or Fe(III) as electron acceptor. Only D. metallireducens completely lacked this ability. Up to 18 putative demethylase operons were identified in the genomes of desulfitobacteria. Bacterial enrichment cultures that originated from five different topsoils sampled in the vicinity of Jena (Germany) were used to assess desulfitobacterial O-demethylation in anoxic consortia. Enrichment with syringate and thiosulfate triggered an increase in Desulfitobacterium-specific 16S rRNA genes in all cultures as confirmed via quantitative PCR. The enrichment was followed by a loss of desulfitobacterial gene copies over time. Community-scale sequencing suggested an outcompetion of desulfitobacteria by acetogens. To allow a better quantification of desulfitobacteria in environmental samples, a new qPCR assay was established that uses the Desulfitobacterium-specific formyltetrahydrofolate synthetase (FTHFS) gene as marker. In contrast to the 16S rRNA gene assay, which detects several gene copies per genome, the new FTHFS assay detects only one gene copy. It is concluded that Desulfitobacterium spp. are involved in the O-demethylation of phenyl methyl ethers in the environment and play an important role within the network of lignin degradation in soils.