Spatially variable areas, or hotspots, of elevated mercury (Hg) concentrations in soil, water, and wildlife occur throughout the Everglades wetland ecosystem. This study investigates the stoichiometric controls of Hg relative to soil, water, and biotic components. Surface water, porewater, soil, periphyton, and Gambusia spp. (mosquitofish) were collected from hotspots and non-spot stations and analyzed for various parameters, including total mercury (THg), organic carbon (OC), total carbon (TC), total phosphorus (TP), and total nitrogen (TN) between late 2010 and early 2013. Soil nutrient ratios were significantly different between hotspot and non-hotspot stations, indicating a difference in trophic status and position along the decay continuum or differences in limiting nutrients. Overall, soil total Hg concentrations were negatively correlated with soil TC/TN, while soil TC/TP and soil TN/TP molar ratios and soil THg were negatively correlated at hotspot stations. Meanwhile, mosquitofish THg was negatively correlated with soil TC/TN molar ratio and positively correlated with soil TC/TP and TN/TP molar ratios, suggesting trophic truncation. Soil, surface water, and porewater THg, TC, and OC interactions resulted in significant differences between hotspot and non-hotspot stations and between molar ratios of C, N, and P. Periphyton-surface water THg/OC homeostasis and soil nutrient ratios significantly explained mosquitofish THg concentrations, further indicating a trophic influence on mosquitofish THg and potential hotspot dynamics. Several factors and processes including bottom-up trophic interaction and vegetation influence on Hg accumulation dynamics and food-chain length explain the development and persistence of Hg hotspot formation within the Everglades system.