Mercury bioaccumulation in aquatic biota poses a widespread threat to human and environmental health. Methylmercury (MeHg), the toxic form of mercury, tends to build up under anaerobic conditions in the profundal zones of lakes. In this study we performed a two-year assessment of spatial and temporal patterns of dissolved oxygen, nitrate, MeHg, manganese (Mn) and iron (Fe) in Occoquan Reservoir, a large run-of-the-river drinking water reservoir in Virginia, USA. A tributary to the reservoir receives input of nitrate-rich tertiary-treated wastewater that enhances the oxidant capacity of bottom water. Multiple lines of evidence supported the hypothesis that the presences of nitrate and/or oxygen in bottom water correlated with low MeHg in bottom water. Bottom water MeHg was significantly lower in a nitrate-rich tributary (annual mean of 0.05 ng/L in both 2012 and 2013) compared to a nitrate-poor tributary (annual mean of 0.58 ng/L in 2012 and 0.21 ng/L in 2013). The presence of nitrate and oxygen in bottom water corresponded with significantly lower bottom water MeHg at an upstream station in the main reservoir (0.05 versus 0.11 ng/L in 2013). In 2012 the reservoir exhibited a longitudinal gradient with nitrate and oxygen decreasing and MeHg and Mn increasing downstream. In both study years, there was a clear threshold of oxygen equivalent (3–5 mg/L), a metric that combines the oxidant capacity of nitrate and oxygen, above which MeHg (<0.05 ng/L), Mn (<0.3 mg/L) and Fe (<0.5 mg/L) were low. Results indicated that the addition of nitrate-rich tertiary-treated wastewater to the bottom of anaerobic reservoirs can reduce MeHg concentrations, and potentially decrease mercury bioaccumulation, while increasing the safe water yield for potable use.