This study reported a novel observation that the long-term cultivation of sulfur-reducing bacteria (S0RB) from a sulfate-reducing bacteria (SRB)-abundant seeding sludge with elemental sulfur feeding significantly shaped the microbial community structure and eliminated the mercury methylation potential in the S0RB-enriched sludge. In this study, the enrichments of SRB and S0RB from activated sludge were obtained through long-term cultivations. Subsequently, the batch tests showed that approximately 5000 μg/L Hg (II) was completely removed from the solution by both the SRB-enriched and S0RB-enriched sludge. Extremely low or no MeHg production was observed in the S0RB-enriched sludge (less than the limit of detection, 0.01 μg/L), while 1.49 μg/L MeHg accumulated in the SRB-enriched sludge. Other batch tests using the sludge samples from a replication of the cultivation showed that the methylation capability of the S0RB-enriching sludge gradually diminished to a negligible level over a 6 month cultivation time. However, some mercury-methylation-related bacteria were present in the enrichment of S0RB such as Geobacter. The absence of MeHg in the S0RB-enriched sludge may be attributed to the dissolved organic matter (DOM) instead of the sulfur- and sulfate-reduction pathway or MeHg demethylation when exposed to Hg (II). The cultivated S0RB could be used for mercury-contaminated wastewater treatment without MeHg concern.