Abstract: Listeria monocytogenes and Vibrio parahaemolyticus are common foodborne pathogens, both of which are capable of forming biofilms. The quorum sensing (QS) system plays an important role in regulating bacterial biofilm formation; therefore, interfering with the QS system using QS inhibitors (QSIs) provides an important target for the prevention and control of bacterial biofilms. In this study, an active substance Z074 extract with targeted quenching activity against QS signal molecules was obtained from Lactiplantibacillus plantarum Z074. The effects of the Z074 extract on biofilm formation, mature biofilm eradication rate, autoinducer-2 (AI-2) activity, and extracellular polysaccharide (EPS) synthesis were investigated, and its regulatory effects on the mixed biofilms of the two bacteria were explored. The results showed that the minimum inhibitory concentration (MIC) of the Z074 extract against the mixed bacterial culture was 3.2 mg/mL. After treatment with 1.6 mg/mL of the Z074 extract for 12 h, the formation amounts of single-species and mixed biofilms of L. monocytogenes and V. parahaemolyticus decreased by 61.82%, 62.30%, and 65.30%, respectively. The eradication rate on the mature mixed biofilm reached 67.54% after treatment with 12.8 mg/mL of the Z074 extract for 4 h, and the eradication rates against the single-species biofilms of the two bacteria were 68.36% and 68.72%, respectively. The inhibition rates of 1.6 mg/mL of the Z074 extract on AI-2 activity and EPS synthesis in single-species and mixed biofilms were all above 60%. In conclusion, the Z074 extract can inhibit AI-2 activity and EPS production in mixed biofilms, and exhibits inhibitory and eradicative effects on mixed biofilms.