The Northeast Stream Quality Assessment (NESQA) performed mercury (Hg) isotope analysis of prey fish, game fish, and sediments from 29 sites across an urban to forested land-use gradient. The data presented here includes the chemical analysis of Hg concentrations and isotopes, capture information for fish species, land use, and stream water quality variables. Using these parameters it was determined that the preservation of Hg isotope signatures, which are indicative of source (e.g. atmospheric deposition, industrial point discharge), were dependent on land use, proximity to point sources, and variables related urbanization (e.g. road density and impervious surface cover). Additional Information regarding the interpretation of this data set can be found in the corresponding journal article: (https://pubs.acs.org/doi/10.1021/acs.est.9b03394).