Chalcopyrite dissolution was evaluated by bioleaching and electrochemical experiments with thermophileA. manzaensis (Acidianus manzaensis) and mesophileL. ferriphilum (Leptospirillum ferriphium) cultures at 65 °C and 40 °C, respectively. It was investigated that the bioleaching of chalcopyrite was stepwise. It was reduced to Cu2S at a lower redox potential locating in the whole bioleaching process byA. manzaensisat high temperature while only at initial days of bioleaching byL. ferriphilum at a relative low temperature. No reduced product was detected when the redox potential was beyond a high level (e.g., 550 mV (vs SCE)) bioleached byL. ferriphilum. Chalcopyrite bioleaching efficiency was substantially improved bioleached byA. manaensis compared to that byL. ferriphilum, which was mainly attributed to the reduction reaction occurring during bioleaching. The reductive intermediate Cu2S was more amenable to oxidation than chalcopyrite, causing enhanced copper extraction.