Excessive biomass accumulation is one main problem which will deteriorate the performance stability of biofilters during long-term operation. In order to systematically explore the feasibility of using ozone to control biomass accumulation within biofilters, the influence of ozone on toluene removal performance, pressure drop through filter bed and porosity, biomass growth and carbon balance of biofilters was examined. The results showed adding ozone of concentration no more than 220 mg/m3 wouldn’t adversely affect toluene removal capacity of biofilters. When the inlet ozone concentration achieved 180~220 mg/m3, ozone could significantly control excessive biomass accumulation, improve the filter bed structure and stabilize the pressure drop through filter bed. Furthermore, accelerating the mineralization rate of toluene was the dominant way of biomass accumulation control by ozone.