Agitated bacterial tank bioleaching reactors are currently sparged with air to satisfy both oxygen and CO2 requirements of microbial cells. Under high-sulphide loading conditions, as is the case with high-grade metal sulphide concentrates, the microbial and chemical demand for oxygen is significantly increased during the bioleaching process. Sparging with enriched oxygen gas may offer an alternative process option to increased agitation and sparged aeration, to overcome the mass transfer difficulties at elevated temperatures where thermophilic Archaea, rather than Bacteria, are used. In the case of air sparging, the DO (dissolved oxygen) concentration in tank reactors could not be increased to a point where it would become inhibitory due to the limited oxygen content of air (20.9% O2). The use of enriched oxygen in such reactors at large scale does, however, pose its own set of process risks. The first aim of this investigation was, therefore, to determine the effects of various DO concentrations, in both the limiting and inhibitory ranges, on the microbial activity of Sulfolobus sp. U40813, a typical thermophilic mineral-leaching archaeon. Secondly, the effect of CO2 concentration on the rate of ferrous iron oxidation was investigated. Both the oxygen and CO2 kinetics were examined in controlled batch cultures at 78°C, using ferrous sulphate and potassium tetrathionate as energy sources. The optimal DO concentration for iron oxidation was found to be between 1.5 and 4.1 mg·l−1. The use of elevated DO concentrations (above 4.1 mg·l−1) inhibited the ferrous oxidation rates. The optimal gas CO2 concentration for ferrous iron oxidation was found to be in the range 7–17% (v/v). The iron oxidation rates were, however, severely limited at CO2 concentrations less than 7%, indicating that the CO2 supply was limiting in this range and inhibited the microbial growth rate.