Marine food webs are important links between Hg in the environment and human exposure via consumption of fish. Estuaries contain sediment repositories of Hg and are also critical habitat for marine fish and shellfish species consumed by humans. MeHg biotransfers from sites of production in estuarine sediments to higher trophic levels via both benthic and pelagic pathways. In this study, we investigated the potential for Hg biotransfer to estuarine food webs across a Hg contamination gradient in the Gulf of Maine. Despite the variation in sediment Hg concentrations across sites (>100 fold), Hg concentrations in biota ranged by only 2−4 fold for each species across sites. Sediment contamination alone explained some variation in Hg and MeHg concentrations in biota across sites. However, biogeochemical and ecological factors also explained significant variation in Hg bioaccumulation across species. Contaminated sites had higher total organic carbon concentrations in sediments, which related to a decrease in Hg bioaccumulation (measured as biota−sediment concentration factors). Moreover, concentrations of MeHg were higher in pelagic-feeding than benthic-feeding fauna (determined from δ¹³C), indicating the importance of pelagic pathways in transferring MeHg. Lastly, the proportion of total Hg as MeHg increased with trophic level (measured as δ¹⁵N). These results reveal the importance of both biogeochemical and ecological factors in determining the bioavailability and trophic transfer of MeHg in estuarine food webs.