Acid mine drainage (AMD) is one of the challenging environmental issues in sulfidic mines. These hazardous solutions generally contain a mixture of indigenous iron- and sulfur-oxidizing microorganisms that could be used as a source for biotechnological purposes. In this study, the ability of an AMD from a sulfide-bearing gold mine to biooxidize its high-grade pyritic gold ore was investigated and its efficiency was compared with iron- and sulfur-oxidizing microorganisms from a microbial culture bank. Experiments were conducted at 35 and 45 ̊C, initial pH values of 1.5 and 2 in a Norris culture medium prepared from deionized and saline local waters. The effects of some critical parameters including the initial pH and the concentrations of ferrous or ferric sulfate were investigated on the efficiency of the biooxidation process and gold extraction. The results showed that the AMD microorganisms had a greater ability to oxidize the sulfide ore than the microorganisms from the microbial bank. The addition of ferrous and ferric sulfates increased the efficiency of biooxidation, while high concentrations of these ions caused the formation of inhibitory precipitates (jarosite) and decreased gold extraction. The results showed that biooxidation using the AMD medium in the saline local water increased the extraction of gold from 73 to 99%. It can be concluded that the application of AMD for the treatment of refractory gold sulfide ores could be an efficient solution for increasing gold extraction and reducing environmental problems.