Abstract: In order to clarify the correlation between reactive oxygen species (ROS) and Bacillus cereus biofilms, the ROS mediated by NADPH oxidase in the Bacillus cereus that were isolated from commercially available raw milk was used as the main target. The relationship between the formation of Bacillus cereus biofilms and ROS were analyzed by using supplement hydrogen peroxide (H₂O₂) as exogenous reactive oxygen species (ROS), N-acetylcysteine (NAC) as ROS scavenging agent and diphenyl iodide chloride (DPI) as NADPH oxidase inhibitor. The results showed that the treatment groups of 0.01 μmol/L H₂O₂, 1 μmol/L H₂O₂ and 100 μmol/L H₂O₂ average biofilms were higher than the control group average biofilms (P<0.05). There was no significant difference between the treatment groups of 0.1 μmol/L H₂O₂ and 10 μmol/L H₂O₂ and the control group average biofilms (P>0.05). With the increase of H₂O₂ concentration, ROS gradually decreased. The treatment groups of NAC average biofilms were higher than the control group average biofilms (P<0.05). ROS decreased gradually with the increase of NAC concentration. When DPI concentration≤1 μmol/L, with the increase of DPI concentration, the average biofilms were higher than the control group average biofilms (P<0.05), and ROS gradually decreased. When DPI concentration>5 μmol/L, with the increase of DPI concentration, the average biofilms were lower than the control group average biofilms (P<0.05), and the ROS increased. The results showed that without affecting the growth and activity of Bacillus cereus, the reduction of ROS induced by certain concentration of H₂O₂, NAC and DPI enhanced the Bacillus cereus biofilms. Confocal laser scanning microscopy showed stronger staining and red network structures in the treated groups compared to the control groups, while the untreated groups showed loose structures and fewer biofilms, suggesting that ROS inhibits the formation of Bacillus cereus biofilms.