Mercury (Hg) and its organic form, monomethylmercury (MeHg), pose potent risks to both ecological and human health in freshwater ecosystems across the globe. Human activities have played a large role in releasing Hg into natural environments, where it can be transformed into MeHg primarily through microbial pathways. Within lotic systems, evidence suggests that benthic algal biofilms known as periphyton are hotspots for MeHg production. Periphyton are composed of complex microbial communities of prokaryotes and autotrophic and heterotrophic eukaryotes that form the base of many aquatic food webs, which may contribute to the bioaccumulation of MeHg at higher trophic levels. It is therefore important to understand how the composition of these periphyton assemblages vary and how such variation may influence Hg methylation and trophic transfer within the ecosystem. Here we present findings from field experiments conducted in East Fork Poplar Creek (EFPC), a 1st to 4th Strahler order stream on the Oak Ridge Reservation (ORR) in East Tennessee, USA. The ORR is a 33,476-acre Federal property with a long history of anthropogenic impact from the Manhattan project and other industrial activities, including discharges of Hg into the environment. Periphyton settlement arrays were deployed quarterly over a one-year period at two sites within EFPC, up- and downstream of a wastewater treatment facility outfall. Periphyton assemblages were sampled after six and 12 weeks of in-stream growth and analyzed for chlorophyll and MeHg content and community metabolism. DNA amplicon sequencing was performed using primers for three different genes targeting assemblages of prokaryotes (16S rRNA), eukaryotes (18S rRNA), and mercury methylators (hgcAB). Findings indicate seasonal and spatial differences in concentrations of chlorophyll and MeHg, and community metabolism within the periphyton assemblages. However, while the prokaryote and eukaryote periphyton assemblages exhibited both seasonal and spatial variation, mercury methylator assemblages did not. The implications of these findings and future areas of research will be discussed.