Increased lake eutrophication, influenced by changing climate and land use, alters aquatic cycling and bioaccumulation of mercury (Hg). Additionally, seasonally dynamic lake circulation and plankton community composition can confound our ability to predict changes in biological Hg concentrations and sources. To assess temporal variation, we examined seasonal total Hg (THg) and methylmercury (MeHg) concentrations and stable isotope values in seston, waters, sediments, and fish from two adjacent urban eutrophic lakes in Madison, Wisconsin. In Lake Monona, surface sediment THg concentrations were elevated due to comparably higher urbanization and historical industrial inputs, whereas Lake Mendota sediments had lower concentrations corresponding with largely agricultural and suburban surrounding watershed. Surface water THg and MeHg were similar between lakes and seasonally dynamic, but water profiles exhibited elevated concentrations in the meta- and hypolimnion, highlighting water column MeHg production. Seston MeHg concentrations were often highest at shoulder seasons, possibly owing to metalimnetic MeHg delivery, but also differences in biomass and water clarity. The ∆199Hg and δ202Hg values in seston were similar between lakes, despite differing sediment THg concentrations and values, suggesting a shared bioaccumulated source of MeHg. Measurement of MeHg stable isotopes further elucidated that seston and fish predominantly bioaccumulated pelagic-sourced MeHg.