This study examines the effects of several operating parameters on copper leaching from chalcopyrite ores using an adapted mesophilic bacterial culture. Three temperatures (35, 40, and 45°C), three pulp density (1, 2, and 4% (w/v)), and three initial ferrous ion (Fe(II)) concentrations (5, 10, and 20 g/L) were employed as variable parameters, and their effects on the bioleaching efficiency of chalcopyrite were investigated. After 14 days, the maximum copper bioleaching efficiency was estimated to be ∼64% at a temperature of 45°C, a pH of 1.5, an initial ferrous concentration of 5 g/L, and a pulp density of 4%. More specifically, the chalcopyrite dissolution tests conducted at different temperatures showed a minimal effect of temperature and low leaching efficiency (<20%) regardless of temperature. The trend of chalcopyrite dissolution at different pulp densities showed that Cu extraction tended to increase with increases in pulp density. Moreover, the Cu leaching efficiency associated with mesophilic microorganisms largely decreased when the initial Fe(II) concentration was greater than 10 g/L. The Cu leaching behavior in different test conditions was evalauted with concentrations of total iron (Fe), Fe(II), and ferric ions (Fe(III)), as well as the oxidation-reduction potential (ORP) of the solution used in the test. The Cu leaching rate increased under lower ORP conditions, lower Fe(III):Fe(II) ratios, and balanced Fe(II)–Fe(III) cycles.