Sulfidic hot springs harbor unique microbial communities and are important in mercury (Hg) species transformations, although the fine scale drivers of these processes remain poorly understood. Here we studied Hg speciation in water, biofilms, and sediment across three sampling seasons in a French sulfidic hot spring with low Hg concentrations. Microbial Hg species methylation and demethylation potentials were evaluated using incubation experiments with species-specific Hg isotope tracers. Temporal variation in inorganic Hg (iHg) and methylmercury (MeHg) concentrations in water, biofilm, and sediment was observed. The incubation of microbial communities in biofilms and sediment under dark conditions exhibited low iHg methylation potentials, whereas a significant extent of biotic MeHg demethylation to oxidized iHg was found in relation to MeHg concentrations. Results from microbial diversity (16S rDNA) and the metabolic inhibition experiments suggest an important role of sulfur-linked bacterial metabolism dynamics. Specifically, sulfate-reducers and anoxygenic phototrophs were important factors in the regulation of MeHg concentrations in our study site. Overall, the observed dominance of microbial MeHg demethylation demonstrates a strong Hg detoxification capacity in sulfidic aquatic environments.