Mercury (Hg) concentrations and isotopic compositions in a range of sample types collected from the legacy Abbadia San Salvatore Mine (ASSM) area were used to evaluate the distribution of Hg in the region. The district generated more than 100,000 metric tons of Hg releasing extensive amounts of gaseous Hg emissions and producing large amounts of mine waste calcine from which Hg can be mobilized into the local and regional environments. Direct and indirect impact from cinnabar ore mining and processing resulted in elevated Hg concentrations in the calcine, soils, lake and stream sediments, and fish samples collected at and downstream of ASSM. The contribution of Hg from the different sources and processes resulted in a wide range of 202Hg and 199Hg isotopic compositions (-2.25 to 0.96‰, -0.12 to 0.72‰ respectively). Fish samples resulted in negative values for 202Hg (-0.53 to -1.21‰) and positive Hg (0.12 to 0.73‰). Primary cinnabar ore and present-day geothermal water and precipitate showed distinctly negative 202Hg (-0.96 to -2.25‰), whereas waste calcine were enriched in 202Hg (202Hg from about -1 to +1‰); soils and sediments show intermediate compositions, reflecting different extents of contributions from the various Hg sources and processes. These sources and processes of Hg include weathering cinnabar, gaseous Hg emissions from ore processing, and geothermal activity to a lesser extent. Hg speciation in concert with Hg isotope ratios can better ascertain source attribution and assist in identifying Hg pathways into the environment to distinguish the more bioavailable forms of Hg entering the ecosystem.