Abstract: To study the inhibitory effects of Enterococcus faecalis Z096 on the biofilm and quorum sensing (QS) system of Vibrio parahaemolyticus, the competition, elimination and exclusion were performed to simulate the interaction between Z096 and V. parahaemolyticus in microbial communities. In addition, the effects of Z096 extract (Z096-E) on V. parahaemolyticus biofilm formation, mature biofilm elimination, cell surface hydrophobicity, auto-aggregation, QS signal molecule AI-2 activity, motility ability (swarming and swimming), extracellular polysaccharide and protein synthesis were further explored. The results showed that Z096 could significantly reduce the number of V. parahaemolyticus cells in plankton and biofilm by competition, elimination and exclusion, and interfere with the adhesion of V. parahaemolyticus on the carrier surface. Moreover, Z096-E could significantly inhibit biofilm formation and effectively eliminate the mature biofilm of V. parahaemolyticus. When treated with 1.6 mg/mL of Z096-E for 12 h, the biofilm inhibition rate was 70.43%, and the metabolic activity decreased by 84.15%; When the mature biofilm of V. parahaemolyticus was treated with 12.8 mg/mL of Z096-E for 4 h, the biofilm removing rate was 58.21%, and the metabolic activity decreased by 69.84%. The swarming and swimming ability, cell surface hydrophobicity and auto-aggregation, extracellular polysaccharide and protein synthesis of V. parahaemolyticus were inhibited by 1.6 mg/mL of Z096-E by 47.26%, 53.56%, 63.37%, 89.38%, 77.65% and 51.91%, respectively, and the inhibitory effect was dose-dependent. In addition, Z096-E weakened the activity of QS signal molecule AI-2 of V. parahaemolyticus, indicating that Z096-E was an AI-2 quorum sensing inhibitor, which could affect the physiological characteristics of V. parahaemolyticus by interfering with the QS system. Therefore, we found one strain of lactic acid bacteria (LAB) that could inhibit V. parahaemolyticus biofilm, and the Z096-E could be used as a novel LAB-based biological agent to prevent and control V. parahaemolyticus biofilm. This study would be of positive significance to eliminate V. parahaemolyticus biofilm pollution and develop novel antibacterial agents.