This dataset details mercury concentrations and mercury stable isotope values collected as part of an environmental mercury assessment encompassing the Mobile and Tombigbee Rivers as well as two U.S. Environmental Protection Agency Superfund sites. The Tensaw River was also sampled as part of this study as a reference site, absent of mercury contamination. As a part of this survey, sediments, waters, and biota (largemouth bass) were measured to assess mercury transport and bioaccumulation of mercury within the Mobile River Basin. High mercury concentrations and isotope values indicative of industrial contamination were found in sediments and fish of Superfund sites. Lower concentrations and isotope values indicative of deposition were found within the Tensaw River and main branches of the Mobile and Tombigbee Rivers.