Abstract: To investigate the effect of phenyllactic acid (PLA) on Listeria monocytogenes biofilms, crystal violet method was used to measure the biomass of L. monocytogenes biofilms, and inverted microscope and scanning electron microscope were used to observe the structure of L. monocytogenes biofilms, the content of extracellular polysaccharide, extracellular protein and extracellular DNA in biofilm was measured by phenol-sulfuric acid method, Folin-phenol method and diphenylamine method, respectively. The effect of PLA on the transcription of genes associated with biofilm formation of L. monocytogenes was detected by real-time quantitative PCR. The results showed that the minimum inhibitory concentration values of PLA against eight L. monocytogenes strains were 6 mg/mL. PLA inhibited L. monocytogenes biofilm formation by reducing the synthesis of extracellular polysaccharides and extracellular proteins. For mature biofilms of L. monocytogenes, PLA destroyed the biofilm structure and reduced the biofilm biomass by reducing the amount of extracellular polysaccharides and extracellular proteins. PLA also inhibited the swarming motility of L. monocytogenes. The transcription levels of quorum sensing system agr in L. monocytogenes were significantly (P<0.05) decreased in the presence of PLA. As a natural antimicrobial compound, PLA had the potential application to control food contamination by L. monocytogenes and its biofilms.