To compare oxidative dissolution rates of chalcopyrite by different consortia of moderately thermophilic acidophiles, various defined mixed cultures of three bacteria Acidithiobacillus caldus s2, Leptospirillum ferriphilum YSK, and Sulfobacillus sp. LN and one archaeon Ferroplasma thermophilum L1 were studied in batch shake flask cultures incubated at 45 °C. Chalcopyrite dissolution was determined by measuring variations of soluble copper, ferric iron, and pH. Microbial population dynamics involved in bioleaching process were monitored using real-time quantitative polymerase chain reaction (PCR) technology. The complex consortia containing both chemoautotrophic (L. ferriphilum and At. caldus) and chemomixotrophic (Sulfobacillus LN and F. thermophilum) moderate thermophiles were found to be the most efficient in all of those tested. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, involving transformations of iron and sulfur and transfer of organic compound, were considered to play a critical role in promoting chalcopyrite dissolution. The real-time PCR assay was reliable to analyze population dynamics of moderate thermophiles in bioleaching systems, and the analysis results were consistent with physiological characteristics of these strains.