The extraction of zinc from low-grade sulfide ores with a complexing in mineralogy have been considered due to the increase the price and high demand for this metal. Among hydrometallurgy processes, bioleaching has been attracted the researcher’s attention because of its advantages such as low cost, environmental friendly, and selectivity of the process. The aim of this research is to investigate the effect of pyrite content on the efficiently of zinc bioleaching process from sulfide sources. In order to this aim, the effect of pyrite content, type of microorganism, pulp density, and mechanical activation on the extraction of zinc from the mentioned source was evaluated using shaking flasks and a Continuous-Stirred Tank Reactor (CSTR). In addition, the effect of the mentioned parameters on the electrochemical behavior of the sphalerite concentrate was studied in the same conditions as the bioleaching environment to understand the process mechanisms and the effect of the parameters. The bioleaching experiments were performed in shake flasks at initial of pH 1.7, pulp density of 5, and 10% (w/v), Norris nutrient medium, stirry rate of 150 rpm at 45 o C . Also, the bioleaching experiments in the CSTR was carried out at 520 rpm, 45 o C, pulp density of 10% (w/v). The results showed that increasing the pyrite content increased the zinc extraction. In shake flasks the maximum zinc recovery of 72% was obtained at 5% pulp density, in the presence of moderate thermophilic microorganisms, and the ratio of sphalerite concentrate to pyrite 50:50 (%wt) maximum zinc extraction in in CSTR way achieved on the pyritic concentrate including 19%iron and 25% zinc. By re-grinding the concentrate, the extraction of zinc was increased in shake flasks. Adding 50% pyrite content to the concentrate caused 77% of the extraction for the activated material probably as a result of increasing of the surface area and instability of the mineral structure. Results showed that the increase of pulp density from 5 to 10% (w/v) caused a decrease in zinc extraction from 49 to 24.5%, which the possible reason for this decrease might be the negative effect of increasing shear stress on the activity of microorganisms and low oxygen mass transfer efficiency in shaking flasks. Also, the results of electrochemical analyses showed that increasing the temperature and re-grinding using planet mill increased the anodic oxidation rate. The results of this research indicated the high potential of the bioleaching in the extraction of zinc from complex iron-bearing concentrate.