Abstract: To determine the efficacy of chalcopyrite bioleaching using pure cultures of Thiobacillus ferrooxidans or Thiobacillus thiooxidans and a mixed culture composed of Thiobacillus ferrooxidans and Thiobacillus thiooxidans, experiments were carried out in shake flasks with Fe²⁺ 4 g·L^-1 and S 1 g·L^-1 at pH=1.80, 130 r/min and 30℃. The tests showed that the copper extraction in a mixed culture composed of Thiobacillus ferrooxidans and Thiobacillus thiooxidans is higher than that in a pure culture. On the other hand, an important potential of Thiobacillus thiooxidans to leaching chalcopyrite was indicated. Thiobacillus thiooxidans can prevent jarosites accumulating on the substrate and allow further copper to dissolute through the action of ferric ion. The selection of the suitable pH in a leaching solution would be significant. Thiobacillus thiooxidans and Thiobacillus ferrooxidans play an important role in the bioleaching process. Finally, the mechanism and the reason for iron precipitation were also discussed in detail.