With the reducing of water resources, using advanced treated refinery wastewater as recirculating cooling water is an effective method to save water and to reduce the pollution of petroleum and petrochemical industry. However, the control of biofilm is a bottleneck in the application of this technology. To resolve the problem of biofilm formation and development, antimicrobial characteristics of chlorine dioxide and benzyldimethyldodecyl-ammonium chloride on biofilm at different substrate levels were investigated. Biofilm detachment ratio and TTC-dehydrogenase activity (DHA) were two indexes to discuss the antimicrobial effects. The results showed that at the high substrate level, the biofilms characteristics (biomass, the content of protein, polysaccharide and EPS) were the higher than those at the medium and low substrate levels, however biofilm's DHA at the medium substrate level (12.97 lgTF/(g h)) was higher than those at the medium substrate level (7.64 lgTF/(g h)) and low substrate level (1.94 lgTF/(g h). The difference of substrate level in the media resulted in different biofilm structure. By contrast with the control experiment, biofilm detachment ratios were all increased in three media with ClO 2 and BDMDAC addition. After ClO 2 addition, MITs were 30, 120 and 240 min and MIC was 1, 4 and 6 mg/L, respectively, at the low, medium and high substrate level. After BDMDAC addition, MITs in three media were all longer than those after ClO 2 addition, MIC was 200, 300 and 400 mg/L, respectively, at the low, medium and high substrate level.