Biooxidation of sulphidic minerals is an important method for extracting gold from refractory gold ores. Biological methods have apparent technical, economical and environmental advantages for extracting metals from low grade ores. The aim of this research is investigating the effects of different chemical and operating factors on Zarshouran gold flotation concentrate, in the presence of mixed culture of mesophilic and moderate thermophilic microorganisms for increment of gold recovery from cyanidation experiment. Special mineralogy of Zarshouran gold ore has made this ore very unique. Entrapping gold in pyrite-arsenopyrite lattice is the reason of refractoriness of gold in this ore. Initial grade of the gold of Zarshouran flotation concentrate was 5.3 ppm. The bioleaching of arsenical gold-bearing sulphidic ores and concentrates extracts Iron, Arsenic and sulphur. Sulphur, Iron, and Arsenic contents of the ore were 1.293%, 4.354% and 0.98%, respectively. For this purpose, after adaptation tests, experiments were done in three parts: biooxidation with mesophilic microorganisms in shaking flask, biooxidation with moderate thermophilic microorganisms in shaking flask and biooxidation with moderate thermophilic microorganisms in agitated rector. Experiments in shake flasks were done by using full factorial design by the use of Design Expert 7.1.3 Software. Four factors were investigated in 2 levels. In experiments with mesophilic microorganisms in shake flasks, nutrient medium (M-9K and Norris), initial pH (1.6 and 2.1), pulp density (5% and 10% (w/v)) and time of biooxidation (10 and 20 days) were investigated. Optimum conditions for the most Iron extraction of 42.12% was predicted in M-9K nutrient medium, pH=1.85 with 5% (w/v) pulp density for 10 days. Optimum conditions for the most Arsenic extraction of 23.95% was predicted in Norris nutrient medium, pH 1.6 with 10% (w/v) pulp density for 20 days. Finally 79% decreasing in sulphur content led to 54% increment in gold recovery from cyanidation tests. In experiments with thermophilic microorganisms in shake flasks, nutrient medium (M-9K and Norris), initial pH (1.6 and 2.1), pulp density (5% and 10% (w/v)) and time of biooxidation (10 and 20 days) were investigated. Optimum conditions for the most Iron extraction of 26.41% was predicted in M-9K nutrient medium, pH=1.6 with 5% (w/v) pulp density for 15 days. Optimum conditions for the most Arsenic extraction of 48.89% was predicted in M-9K nutrient medium and pH=1.6 with 5% (w/v) pulp density for 15 days. Finally 84% decreasing in sulphur content led to 64% increment in gold recovery from cyanidation tests. Biooxidation experiments in agitated reactor with moderate thermophilic microorganisms using Norris medium and pH 1.8 with 10% (w/v) pulp density showed that, Iron and Arsenic extraction were 3.2% and 2.6%, respectively, at the 11 th day of experiment, and 61% decreasing in sulphur content led to 80% increment in gold recovery. Biooxidation experiments in agitated reactor with moderate thermophilic microorganisms using Norris medium and pH 1.8 with gradual increment of solid to 20% (w/v) pulp density showed that, Iron and Arsenic extraction were 1.3% and 1.1%, respectively, at the 31 th day of experiment, and 53% decreasing in sulphur content led to 65% increment in gold recovery. Key words: Mesophilic microorganisms, Moderate thermophilic microorganisms, Shake flask experiment, Stirred tank reactor, Arsenopyrite, Gold recovery, Zarshouran