The microbial mats responsible for biological desulfurization from biogas in a full-scale anaerobic digester were characterized in terms of their structure, as well as their chemical and microbial properties. Filament-shaped elemental sulfur 100-500 μm in length was shown to cover the mats, which cover the entire headspace of the digester. This is the first report on filamentous sulfur production in a non-marine environment. The results of the analysis of the mats suggest that the key players in the sulfide oxidation and sulfur production in the bio-desulfurization in the headspace of the digester were likely to be two sulfide-oxidizing bacteria (SOB) species related to Halothiobacillus neapolitanus and Sulfurimonas denitrificans, and that the microbial community, cell density, activity for sulfide oxidation varied according to the environmental conditions at the various locations of the mats. Since the water and nutrients necessary for the SOB were provided by the digested sludge droplets deposited on the mats, and our results show that a higher rate of sulfide oxidation occurred with more frequent digested sludge deposition, the habitat of the SOB needs to be made in the lower part of the headspace near the liquid level of the digested sludge to maintain optimal conditions.