The term biomining have been coined to refer to the use of microorganisms in mining processes as in the biooxidation of refractory gold minerals. The biooxidation of refractory gold ores presents similar characteristics when compared with roasting and pressure oxidation. Almost without exception, microbial extraction procedures are more environmentally friendly. The isolated bacteria in this study, were included a variety of oxidizing acidophilic autotrophic iron and sulfur oxidizing that named F.O.C.B and C.L.L.B. Biological oxidation with shaking flask method were done in the presence of 1 gr of the ore milled of Mouteh with a particle diameter of 150 microns (100 mesh) in 9K medium without iron , at 30°C and shaking speed 180 rpm, during the 7 days, during this period ferrous ions assessment were performed by colorimetric method with orthophenantrolin. The results showed that F.O.C.B. bacteria reduced the amount of ferrous ion from 0.63 to 0.015 gr/L and C.L.L.B. bacteria from 0.64 to 0.04 gr/L. Also mineral pyrite was removed after 7 days. This study aimed to Optimization of gold extracting from sulfide ore Mouteh using indigenous bacteria.