X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analyses were carried out to investigate the surface species and interfacial reactions during bioleaching of chalcopyrite by different strains of moderately thermophilic bacteria (45 °C). Results show that monosulfide (CuS), disulfide (S22?), polysulfide (Sn2?), elemental sulfur (S0) and sulfate (SO42?) are the main intermediate species on the surface of chalcopyrite during bioleaching byA. caldus,S. thermosulfidooxidans andL. ferriphilum. The low kinetics of dissolution of chalcopyrite inA. caldus can be mainly attributed to the incomplete dissolution of chalcopyrite and the passivation layer of polysulfide. Polysulfide and jarosite should be mainly responsible for the passivation of chalcopyrite in bioleaching byL. ferriphilumorS. thermosulfidooxidans. However, elemental sulfur should not be the main composition of passivation layer of chalcopyrite during bioleaching.