Studies of metal sulfide oxidation in acid mine drainage (AMD) systems have primarily focused on pyrite oxidation, although acid soluble sulfides (e.g., ZnS) are predominantly responsible for the release of toxic metals. We conducted a series of biological and abiotic laboratory oxidation experiments with pure and Fe-bearing sphalerite (ZnS & Zn_0.88Fe_0.12S), respectively, in order to better understand the effects of sulfide mineralogy and associated biogeochemical controls of oxidation on the resultant δ³⁴S and δ¹⁸O values of the sulfate produced. The minerals were incubated in the presence and absence of Acidithiobacillus ferrooxidans at an initial solution pH of 3 and with water of varying δ¹⁸O values to determine the relative contributions of H₂O-derived and O₂-derived oxygen in the newly formed sulfate. . Experiments were conducted under aerobic and anaerobic conditions using O₂ and Fe(III)_aq as the oxidants, respectively. Aerobic incubations with A. ferrooxidans, and S^o as the sole energy source were also conducted. The δ34SSO4 values from both the biological and abiotic oxidation of ZnS and ZnS_Fe by Fe(III)_aq produced sulfur isotope fractionations (ε34SSO4-ZnS) of up to −2.6‰, suggesting the accumulation of sulfur intermediates during incomplete oxidation of the sulfide. No significant sulfur isotope fractionation was observed from any of the aerobic experiments. Negative sulfur isotope enrichment factors (ε34SSO4-ZnS) in AMD systems could reflect anaerobic, rather than aerobic pathways of oxidation. During the biological and abiotic oxidation of ZnS and ZnS_Fe by Fe(III)_aq all of the sulfate oxygen was derived from water, with measured ε18OSO4-H2O values of 8.2 ± 0.2‰ and 7.5 ± 0.1‰, respectively. Also, during the aerobic oxidation of ZnS_Fe and S^o by A. ferrooxidans, all of the sulfate oxygen was derived from water with similar measured ε18OSO4-H2O values of 8.1 ± 0.1‰ and 8.3 ± 0.3‰, respectively. During biological oxidation of ZnS by O₂, an estimated 8% of sulfate–oxygen was derived from O₂, which is enriched in ¹⁸O relative to water, thus resulting in a larger apparent ε18OSO4-H2O value of 9.5‰. Based on the data presented we hypothesize that the similar ε18OSO4-H2O values of ∼8‰ from all of the aerobic and anaerobic experiments result from a common rate-limiting step that involves oxygen isotopic exchange between a sulfite (SO3-) intermediate and H₂O. Our results indicate that the δ18OSO4 values cannot be used to distinguish biological and abiotic, nor aerobic versus anaerobic, pathways of sphalerite oxidation. However, the ε18OSO4-H2O values of ∼8‰ measured here are distinctly higher than ε18OSO4-H2O values of ∼4‰ previously reported for pyrite oxidation indicating the influence of sulfide mineralogy on measured δ18OSO4 values.