Estuaries are transitional habitats characterized by complex biogeochemical and ecological gradients that result in substantial variation in fish total mercury concentrations (THg). We leveraged these gradients and used carbon (δ13C), nitrogen (δ15N), and sulfur (δ34S) stable isotopes to examine the ecological and biogeochemical processes underlying THg bioaccumulation in fishes from the San Francisco Bay Estuary. We employed a tiered approach that first examined processes influencing variation in fish THg among wetlands, and subsequently examined the roles of habitat and within-wetland processes in generating larger-scale patterns in fish THg. We found that δ34S, an indicator of sulfate reduction and habitat specific-foraging, was correlated with fish THg at all three spatial scales. Over the observed ranges of δ34S, THg concentrations in fish increased by up to 860% within wetlands, 560% among wetlands, and 291% within specific impounded wetland habitats. In contrast, δ13C and δ15N were not correlated with THg among wetlands and were only important in low salinity impounded wetlands, possibly reflecting more diverse food webs in this habitat. Together, our results highlight the key roles of sulfur biogeochemistry and ecology in influencing estuarine fish THg, as well as the importance of fish ecology and habitat in modulating the relationships between biogeochemical processes and Hg bioaccumulation.