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Wastewater sludge is used as an alternative fuel due to its high organic content and calorific value. However, influent characteristics and operational practices of wastewater treatment plants (WWTPs) can increase the sulfur content of sludge, devaluing it as a fuel. Thus, we investigated the biochemical mechanisms that elevate the sulfur content of sludge in a full-scale industrial WWTP receiving wastewater of the textile dyeing industry and a domestic WWTP by monitoring the sulfate, sulfur, and iron contents and the biochemical transformation of sulfate to sulfur in the wastewater and sludge treatment streams. A batch sulfate reduction rate test and microbial 16S rRNA and dsrB gene sequencing analyses were applied to assess the potential and activity of sulfate-reducing bacteria and their effect on sulfur deposition. This study indicated that the primary clarifier and anaerobic digester prominently reduced sulfate concentration through biochemical sulfate reduction and iron–sulfur complexation under anaerobic conditions, from 1247 mg/L in the influent to 6.2~59.8 mg/L in the industrial WWTP and from 46.7 mg/L to 0~0.8 mg/L in the domestic WWTPs. The anaerobic sludge, adapted in the high sulfate concentration of the industrial WWTP, exhibited a two times higher specific sulfate reduction rate (0.13 mg SO42−/gVSS/h) and sulfur content (3.14% DS) than the domestic WWTP sludge. Gene sequencing analysis of the population structure of common microbes and sulfate-reducing bacteria indicated the diversity of microorganisms involved in biochemical sulfate reduction in the sulfur cycle, supporting the data revealed by chemical analysis and batch tests. [ABSTRACT FROM AUTHOR]