Mercury (Hg) is a global contaminant of significant concern, especially due to its transformation into methylmercury (MeHg), a potent neurotoxin. River biofilms, such as those found in the Tapajós River, are recognized as crucial hotspots for microbial methylation of Hg. This study investigated the interaction between Hg and selenium (Se), an element known to mitigate Hg toxicity, in freshwater biofilms. The objective was to evaluate Hg speciation and correlate it with the detection of key genes involved in Hg methylation and resistance (hgcA, merA, gcs, dsrA) under different exposure conditions to IHg and SeO₃^2-. The results demonstrated that biofilms act as efficient sinks for Hg, with high bioaccumulation of the metal. Speciation analysis revealed that Hg remained predominantly in the inorganic form (IHg) in the dissolved phase, with MeHg was below the detection limits, suggesting a low methylation rate under experimental conditions. PCR analysis revealed a detection pattern suggesting strong selectivity and toxicity of IHg. The resistance gene merA was not detected in treatments with IHg alone and IHg+ SeO₃², suggesting the elimination of the subpopulation carrying this gene due to toxicity. Crucially, the methylation gene hgcA, which was detected in single treatments with IHg, was not detected under the co-exposure to IHg + SeO₃^2-, suggesting that SeO₃^2- mitigated the methylation. From an environmental perspective, our results suggest that naturally occurring Se may play a protective role in Amazonian ecosystems impacted by Hg, reducing the bioavailable fraction of this potent neurotoxin and, consequently, eliminating susceptible microbial populations. This has important implications for risk assessment and potential bioremediation strategies in regions affected by artisanal gold mining and other sources of Hg contamination.