In addition to physical and chemical technologies, bioprocesses have been proven to be efficient and cost-effective alternative technologies for the treatment of volatile organic compounds (VOCs) to prevent environmental and health problems. One of the solutions for overcoming the mass transfer limitation of VOCs and improving the biodegradation efficiency is to use non-aqueous phases or surfactants. In this study, the biodegradation of hexane, as a well- known VOC, in the concentration range of 7-55 g/m3 in presence of saponin, sodium dodecyl sulfate (SDS), and silicone oil was investigated, as a non-ionic biosurfactant, an anionic surfactant and a non-aqueous phase, respectively. Initial concentrations of hexane, saponin, sodium dodecyl sulfate, and silicone oil were optimized by response surface method (RSM) to achieve a maximum specific degradation rate (SDR) of hexane. Saponin had a negative effect on hexane (55 mg/L) biodegradation, especially at concentrations >1 CMC, while the optimum concentration was obtained as 0.06 CMC. The degradation of hexane was inhibited by SDS because of its high toxicity on microorganisms. Maximum SDR was observed at 54 g/m3 hexane and 2% v/v silicone oil. It should be mentioned that, average amount of removal efficiency (RE) for hexane in the presence of saponin (REavg.=83%) was more than silicone oil (REavg.=63%). That negative effect of silicone oil on hexane biodegradation could be due to the substrate inhibition for microorganisms. This may be attributed to that hexane possesses a higher solubility in liquid phase in the presence of silicone oil. Therefore, at elevated hexane concentrations, the presence of surfactants and silicone oil may decrease the biodegradation rate due to change of bioavailability of VOC and increasing the risk of toxicity. The correct selection of the additive with optimum concentration, regarding the biomass capability in degrading pollutants, is critical to reach maximum performance.