Bioleaching is an important process in metallurgy and in environmental sciences, either for the acquisition of metals or for the formation of acid rock drainage. In this study the implications of the processes during bioleaching of a pyritic chalcopyrite concentrate were analysed regarding its Cu and Fe isotope fractionation. The development of the redox potential during the bioleaching experiment was then simulated in an electrochemical cell in absence of microorganisms to investigate the effect of microbial activity on the Cu and Fe isotope fractionations. The leaching experiments were performed for 28 days at 45 °C with a solid content of 2.5% (w/v) at pH 1.5. It was found that Cu dissolution efficiency was similar in both experiments and the leaching curves were linear with no sign of passivation due to presence of pyrite. The heavy Cu isotope (δ65Cu) was leached more easily and as a result the leachate was enriched with the heavy Cu isotope at the beginning of both experiments and as the leaching progressed δ65Cu values in the leachate became similar to the ones of the chalcopyrite concentrate, confirming an equilibrium fractionation happening in a closed system. There was no distinct difference in the Cu and Fe isotope fractionations in absence and presence of microorganisms. Finally based on Cu and Fe isotope signatures, a simplified method is suggested for the estimation of the leaching extent during the oxidisation of sulphide materials in natural systems.