To evaluate the effect of iron-based nanozymes on the biofilm of S.typhimurium, the biofilms were detected by crystal violet staining method, biofilm integrity, biofilm biomass, bacterial activity, and the intracellular ROS levels of S.typhimurium S025. Our results showed that, after treatment with iron oxide (Fe₃O₄) nanozyme and iron sulfide (nFeS) nanozyme during biofilm formation for 48 h, the A₅₅₀ value, biofilm height, biofilm biomass, and bacteria viability were significantly decreased compared to the untreated group, but the intracellular ROS levels remarkably increased. Moreover, to determine whether there is a similar inhibitory effect on pre-formed biofilms, Fe3O4 nanozyme and nFeS nanozyme were incubated from the apical side of the biofilms for different time. As expected, both nanozymes appreciably destroyed the biofilms that had formed. Importantly, nFeS nanozyme showed better efficacy than Fe₃O₄ nanozyme in above biofilm treatment. Taken together, these findings clearly demonstrated that iron-based nanozymes, as a suitable agent, regulated intracellular ROS levels to prevent the biofilm formation of S.typhimurium and destroyed the matured biofilm, which is helpful to preventing and treating relevant diseases caused by biofilm of S.typhimurium.