Bio-oxidation of refractory sulfidic gold minerals has been applied at the commercial scale as a pre-treatment to improve gold yields and reduce chemical consumption during gold cyanidation. In this study, the effect of initial cell concentration on the oxidation of pyritic gold ore was evaluated with four aerated bioreactors at 30 °C with 10% pulp density and pH maintained at 1.4 with NaOH. Results of NaOH consumption and changes in soluble Fe and S concentrations indicated that increasing the initial cell concentration from 2.3 × 107 to 2.3 × 1010 cells mL−1 enhanced pyrite oxidation during the first week. However, by day 18 the reactor with the lowest initial cell concentration showed profound performance enhancement based on soluble Fe and S concentrations, sulfide-S and pyrite contents in the residues, and subsequent gold leaching of the bio-oxidation residues by cyanidation. Overall, the results showed that the cell concentration was clearly beneficial during the initial stages of oxidation (first 7–8 days)