Low-concentration ozonation was developed as a novel technique to control the excess biomass in volatile organic compound (VOC) biofilters. In order to understand the reaction mechanism between ozone and biomass, the changes in properties of ozone exposed extracellular polymeric substances (EPS) were investigated in this study. EPS was sequestered from the biofilm, obtained from a biofilter treating gaseous toluene, and then it was exposed to gaseous ozone at 272 ± 22 ppm continuously for 12 h. The total organic carbon (TOC) results indicated that low concentration ozone could not mineralize the EPS to carbon dioxide (CO2) completely. The excitation-emission matrix fluorescence spectroscopy (EEM) results demonstrated that ozone preferred to attack the benzene ring and specific amino acid residues (such as tryptophan) on the protein chain. High performance size-exclusion chromatography (HPSEC) results confirmed that the protein molecules were depolymerized after ozone attack, while the molecular weight of polysaccharides was not much affected by ozone. During ozonation, few volatile organic compounds (VOCs), such as carboxylic acids, aldehydes, ketones, benzaldehyde and by-products of toluene, were generated, which confirms a minor change in the TOC concentration of EPS. Results revealed that low concentration ozone can reduce the molecular weight of biofilter EPS which can be a key reason for controlling biomass accumulation. Additionally, this can be used to study the composition of biofilm EPS from biofilters.