To analyze the impact of the fliE gene on the persistence of Listeria monocytogenes, a fliE deletion mutant was successfully constructed using homologous recombination technology. Growth capacity assays demonstrated that the ΔfliE strain reached the stationary phase at an OD600 of 0.64, whereas the parental strain reached the stationary phase at an OD600 of 0.66. No significant difference in growth capability was observed between the parental and ΔfliE strains (P>0.05). Motility assays demonstrated that the migration diameters of the parental and ΔfliE strains in a semi-solid agar medium were 6.00 ± 0.05 mm and 6.03 ± 0.05 mm, respectively, suggesting no significant difference in motility (P>0.05). The biofilm formation capability of the ΔfliE strain was assessed using the crystal violet staining method. Compared with the parental strain, the ability of the ΔfliE strain to form mixed biofilms with Weissella confusa W4-1 and WL5-1 decreased to 71.55% and 74.77% (P<0.01), respectively. Disinfectant tolerance assays and analysis based on confocal laser scanning microscopy (CLSM) suggested that the survival ability of L. monocytogenes in mixed biofilms formed by co-culturing the ΔfliE strain with Weissella confusa was significantly reduced. These results demonstrated that deletion of the fliE gene not only affected the biofilm formation ability of L. monocytogenes but also reduced its tolerance to quaternary ammonium disinfectants. The findings of this study provide a theoretical foundation for further understanding the molecular mechanisms underlying the persistence of hypervirulent, persistent-genotype L. monocytogenes strains.