Stable isotope (13C,18O,34S) and trace element (Sr2+, Mg2+, Mn2+, Ba2+, Na+) investigations of elemental sulfur, primary calcites and mixtures of aragonite with secondary, post-aragonitic calcite from sulfur-bearing limestones have provided new insights into the geochemistry of the mineral forming environment of the native sulfur deposit at Machów (SE-Poland). The carbon isotopic composition of carbonates (δ13C = −41 to −47‰vs.PDB) associated with native sulfur (δ34S = + 10 to + 15‰vs.V-CDT) relates their formation to the microbiological anaerobic oxidation of methane and the reduction of sulfate derived from Miocene gypsum. From a comparison with experimentally derived fractionation factors the element ratios of the aqueous fluids responsible for carbonate formation are estimated. In agreement with field and laboratory observations, ratios near seawater composition are obtained for primary aragonite, whereas the fluids were relatively enriched in dissolved calcium during the formation of primary and secondary calcites. Based on the oxygen isotope composition of the carbonates (δ18O = −3.9 to −5.9‰vs.PDB) and a secondary SrSO4(δ18O = + 20‰vs.SMOW; δ34S = + 59‰vs.V-CDT), maximum formation temperatures of 35°C (carbonates) and 47°C (celestite) are obtained, in agreement with estimates for West Ukraine sulfur ores. The sulfur isotopic composition of elemental sulfur associated with carbonates points to intense microbial reduction of sulfate derived from Miocene gypsum (δ34S ≈ + 23‰) prior to the re-oxidation of dissolved reduced sulfur species.