In this study, bacterial leaching of zinc, copper and antimony from tetrahedrite concentrate was investigated with mixed mesophilic and mixed moderately thermophilic microorganisms. The mineralogical studies showed that tetrahedrite ((Cu,Fe)12Sb4S13), ankerite (Ca(Fe,Mg)(CO3)2) and quartz (SiO2) are the main mineral phases. During bioleaching experiments, the effects of different chemicals and factors such as graphite, silver nitrate, L-cysteine, pyrite, and mechanical activation were studied for the first time. It was found that the removal of toxic antimony from tetrahedrite concentrate by alkaline Na2S-NaOH facilitates the growth of microorganisms and extraction rates of metals. The maximum extraction of zinc, copper and antimony was 99.7%, >99.9% and 92.8% with ORP, cell counts and Fe(III) concentration of 666 mV, 108 cells/ml and 9.58 g/l, respectively, using the moderately thermophilic culture with the tetrahedrite residue obtained after alkaline leaching (low-Sb). Under the same control leaching conditions with H2SO4, the maximum extraction of zinc, copper and antimony was 26.2%, 46.1% and 10.7% with low ORP and Fe(III) concentration of 464 mV and 5.18 g/l, respectively. The results showed that bacterial leaching of tetrahedrite concentrate significantly improves the extraction of metals compared to control test. As an important result, mechanical activation by planetary mill played a significant role during tetrahedrite bioleaching. Residual analysis proved the formation of antimony sulfide (Sb2S5) and jarosite (K3Fe9S6O42H18) phase as dissolution inhibitors and acanthite phase (Ag2S) as accelerator for dissolution kinetics.