Few published studies present data on relationships between fish mercury and surface or pore water sulfate concentrations, particularly on an ecosystem-wide basis. Resource managers can use these relationships to identify the sulfate conditions that contain fish with health-concerning total mercury (THg) levels and to evaluate the role of sulfate in methyl-mercury (MeHg) production. In this study, we derived relationships between THg in three fish trophic levels (mosquitofish, sunfish, and age-1 largemouth bass) and surface water sulfate from 1998 to 2009 for multiple stations across the Everglades Protection Area (EPA). Results show the relationship between sulfate and fish THg in each fish type is nonlinear and largely skewed, similar to the relationship between MeHg production and sulfate concentration in peatland sediment pore water identified by other researchers. Peak fish THg levels occurred in ~1 to 12 mg/L sulfate conditions. There was significant variability in the fish THg data, and there were several instances of high-fish THg levels in high-sulfate conditions (>30 mg/L). Health-concerning fish THg levels were present in all surface water sulfate conditions; however, most of these levels occurred in 1-20 mg/L sulfate. The data in this study, including recent studies, show consistent and identifiable areas of high- and low-fish THg across the spectrum of surface water sulfate concentration, therefore, applying an ecosystem-wide sulfur strategy may be an effective management approach as it would significantly reduce MeHg risk in the EPA.