A pyrite mineral surface was exposed to a mixed culture of the lithotrophic bacteria Thiobacillus ferrooxidans (Tf). The surface structure of this sample was compared to surfaces exposed to uninoculated solutions of either sulphuric acid or ferric and ferrous sulfate dissolved in sulfuric acid solution at equivalent concentrations as the inoculated solution. All surfaces were examined using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). A chemical profile of the surfaces, as a function of depth, was obtained by surface ablation. The colonized surface produced an oxidized phase extending to a depth of more than 4 μm. A carbonaceous phase was not observed within the oxide layer. No oxide layers of significant depth were observed for samples exposed to uninoculated solutions. The importance of the sessile cells in bio-oxidation of pyrite is demonstrated and a qualitative model is proposed.