Flotation separation of sphalerite from chalcopyrite is significantly affected by the oxidation of metal species on the surface of sphalerite, partially contributed by production of the reactive oxygen species hydrogen peroxide (H2O2) and hydroxyl radicals (OH center dot) if any during wet grinding of a complex sulphide ore. This research experimentally measures the production of H2O2 during grinding of a Cu/Zn ore from Mine Matagami (Canada) with different grinding time and in different grinding environments. Results reveal that H2O2 was formed spontaneously when the ore was ground in the ball mill. The amount of H2O2 generated increases with an increased pyrite load to the ball mill. Grinding with mild steel shows a lower measurable concentration of H2O2 in slurry relative to grinding with stainless steel balls. This appears to be in conflict with other researchers. It is found that Fe ions released from mild steel balls benefits the conversion from H2O2 to (OH center dot), but in the current H2O2 detection program hydroxyl radicals (OH center dot) could not be measured. Surface analysis of mineral grains from the mill discharge samples has identified an obvious correlation between H2O2 detected in the pulp and sphalerite surface oxidation. Lower pulp H2O2 concentrations possibly correspond to the conversion of H2O2 to OH center dot which may be linked to a more pronounced sphalerite surface oxidation.