The data in this data release includes results from the analysis of water and fish from 76 sites in the Everglades National Park (ENP). Water and particulate matter samples were collected from 2008 to 2018 and analyzed for total mercury (THg) and methylmercury (MeHg). Filtered water samples were also analyzed for dissolved organic carbon (DOC), specific ultraviolet absorbance (SUVA), and major anions. Fish samples (Eastern Mosquitofish, Flagfish, African Jewelfish, Golden Topminnow, and Mayan Cichlid species) were collected from 2007 to 2018 and analyzed for THg and MeHg and carbon and nitrogen stable isotopes. These data are important for analyzing hydrologic and geochemical controls on MeHg distribution and production in the ENP.