Recently, ozone injection technique was developed as a novel biomass control method to reduce bed clogging in biofilters treating volatile organic compounds (VOCs). However, the effects of ozone on the characteristics of biofilms are still unknown. In this study, two identical lab-scale biofilters treating gaseous toluene were operated in parallel except that one was continuously injected with 200 mg/m(3) ozone. Four glass slides were placed inside each biofilter on day 57 and then were taken out sequentially after 1, 2, 4, and 6 weeks of cultivation. The biofilms grown on the glass slides were stained by the ViaGram (TM) Red + Bacterial Gram Stain and Viability Kit and observed through the confocal laser scanning microscopy (CLSM). According to the CLSM images of 1, 2, and 4 weeks, the ozonated biofilm was significantly thinner than the control biofilm, which demonstrated that ozone could effectively control the biomass in the biofilter. For the biofilter without ozone injection, the ratios of viable cells (0.51 similar to 0.89) and the ratios of Gram-positive bacteria (0.22 similar to 0.57) both decreased within 4 weeks of cultivation. The CLSM image analysis results also demonstrated that a continuous injection of 200 mg/m(3) ozone was able to significantly enhance the ratio of viable cells to 0.77 similar to 0.97 and allow the dominance of Gram-positive bacteria in the biofilms with the ratio 0.46 similar to 0.88 instead of Gram-negative bacteria. For the 6-week samples, the biofilm thickness of the control system was reduced significantly which indicated the detachment of accumulated biofilms might occur in the samples without ozone.