Mercury (Hg) is a nonessential trace metal that occurs in aquatic environments through both natural and anthropogenic deposition. When mercury undergoes methylation, it is transformed into methylmercury (MeHg), a neurotoxin capable of bioaccumulating in food webs. This process is facilitated by methylating microorganisms (e.g. sulfate-reducing bacteria, iron-reducing bacteria, and methanogens), all of which contain a hgcAB gene pair. High levels of methylmercury have been recorded in local fish populations near run-of-river hydroelectric dams along the St. Maurice River in Québec, Canada. In order to investigate the source MeHg levels, periphyton biofilms were collected from three sampling sites upstream of the dam, a natural, flooded and artificial wetland site. Using periphyton biofilm samples, we compared the microbial community composition within and between periphyton biofilms, assessed the natural diversity of mercury methylating microorganisms within periphyton biofilms, and quantified the abundance of mercury methylating microorganisms at each sampling site in relation to mercury methylation and demethylation. Different rates of mercury methylation were observed in all three sampling sites, the highest rates seen in the artificial wetlands. This was supported by 16S rRNA analyses, which revealed lineages associated with mercury methylating microorganisms being present at all sites, with the greatest abundance observed in the wetlands. Additionally, the presence of mercury methylating microorganisms was later confirmed through the presence of potential hgcA and hgcB genes found within the metagenomes at the wetland sampling site. Overall, these findings highlight the importance of the artificial wetlands and subsequent flooding following the construction of the Chute-Allard hydroelectric generating station, as being important contributors to increased MeHg levels and its bioaccumulation in local fish populations.