Gold is sometimes encapsulated in a matrix of sulphidic minerals such as arsenopyrite and pyrite as submicroscopic grains, which is not extracted efficiently during conventional cyanidation process. On the other hand, worldwide reserves of high-grade ores are diminishing at an alarming rate due to the rapid increase of the demand for metals. It is inevitable that smaller, lower-grade and more complex sulphide resources will need to be processed. Biooxidation of sulphidic minerals is an important method for extracting gold from sulphidic refractory gold ores. Bacterial oxidation has some advantages such as the relative simplicity of the process, mild operational conditions, low entrance energy, more compatible with environment in comparison with physic-chemical processes. In this research, the effect of various factors on the biooxidation of refractory gold ores of Mouteh and Zarshuran gold ores were studied. For this purpose, experiments were performed in five parts: biooxidation of Zarshouran gold ore with mesophilic bacteria in shaking flask, biooxidation of Zarshouran gold ore with moderately thermophilic bacteria in shaking flask, biooxidation of Mouteh gold ore with mesophilic bacteria in shaking flask, biooxidation of Mouteh gold ore with moderately thermophilic bacteria in shaking flask, and biooxidation of Zarshouran gold ore in column rector with 1:1 ratio of mesophilic and moderately thermophilic bacteria. In experiments with both microorganisms and sample ores investigating parameters were; nutrient medium (Norris and 9K), initial pH (1.6 and 2.1), pulp density (5% and 10%) and time of biooxidation (16 and 36 days for Zarshouran ore, 10 and 22 days for Mouteh ore). In biooxidation experiments Zarshouran gold ore with mesophilic bacteria, optimum conditions for the most iron extraction of 48.7% was predicted in pH 1.6 and Norris nutrient medium for 16 days, and optimum conditions for the most arsenic extraction of 35 %was predicted in pH 1.6 and 5%(w/v) pulp density for 36 days. Finally 41.6% reduction in sulphur content led to 76% enhancement in gold recovery after cyanidation. In biooxidation experiments of Zarshouran gold ore with moderately thermophilic bacteria, optimum conditions for the most iron extraction of 26.4% was predicted in pH 1.6 and Norris nutrient medium with 5%(w/v) pulp density, and optimum condition for the most arsenic extraction of 17.5% was predicted in pH 1.6 for 36 days. Finally 80.6% reduction in sulphur content led to 84% enhancement in gold recovery after cyanidation. In biooxidation experiments of Mouteh gold ore with mesophilic bacteria, optimum conditions for the most iron extraction of 55.6% was predicted in 9K nutrient medium with 5%(w/v) pulp density and pH 1.6. Finally 10% reduction in sulphur content led to 69% enhancement in gold recovery after cyanidation. In biooxidation experiments of Mouteh gold ore with moderately thermophilic bacteria, optimum conditions for the most iron extraction of 16% was predicted in pH 1.6 with 9K nutrient medium and 5%(w/v) pulp density. Finally 41.6% reduction in sulphur content led to 82% enhancement in gold recovery from cyanidation. Biooxidation experiments in column reactor with 1:1 ratio of mesophilic and moderately thermophilic bacteria showed that 73% reduction in sulphur content led to 78% enhancement in gold recovery. Key Words: Refractory gold ore, Biooxidation, Arsenopyrite, Pyrite, Shake flask, Column reactor, Zarshouran, Mouteh, Gold recovery