This study investigated the biooxidation of a high-grade refractory gold ore from the Zarshouran mine (West Azarbaijan, Iran) in shake flasks and a stirred tank bioreactor (STBR) using a mixed culture of moderately thermophilic microorganisms. The influence of four critical parameters including, pH, biooxidation time, nutrient medium type and pulp density on the iron and arsenic extraction as well as gold cyanidation were evaluated in a full factorial design in shake flasks at 45 °C. Maximum iron extraction was obtained in M9K medium, pulp density of 5% (w/v), 15 days of biooxidation time and the pH of 1.6. Biooxidation in the STBR was carried out at the pulp densities of 10% and 20% (w/v), the pH of 1.7 in the M9K medium, in which dissolved oxygen, pH, redox potential, iron and arsenic concentrations were measured during the process. It was found that 61% decrement of sulphur content in the STBR led to recover 80% of gold, which was 43% higher than that in the conventional cyanidation process. It can be concluded that the oxidation of refractory arsenopyrite gold ore can achieved using a moderately thermophilic biooxidation culture.