The microbial sulfur removal was investigated on high sulfur content (1.9%) coal concentrate from Tabas coal preparation plant. A mixed culture of ferrooxidans microorganisms was isolated from the tailing dam of the plant. Full factorial method was used to design laboratory test and to evaluate the effects of pH, particle size, iron sulfate concentration, pulp density, and bioleaching time on sulfur reduction. Statistical analyses of experimental data were considered and showed increases of pH and particle size had negative effects on sulfur reduction, whereas increases of pulp density and bioleaching time raised microbial desulfurization rate. According to results of designing, and regarding statistical factors, the optimum values for maximum sulfur reduction were obtained; pH (1.5), particle size (−180 μm), iron sulfate concentration (2.7 mmol/L), pulp density (10%) and bioleaching time (14 d), which leaded to 51.5% reduction from the total sulfur of sample.