Listeria monocytogenes is a common foodborne pathogen associated with listeriosis, a severe illness with a mortality rate of 20–30%. This study aimed to assess the antimicrobial properties of Lactiplantibacillus plantarum TW57-4 cell-free supernatant (CFS) against L. monocytogenes ATCC 19115. The CFS exhibited a minimum inhibitory concentration (MIC) of 31.24 mg/mL against L. monocytogenes. Scanning electron microscopy confirmed its anti-biofilm effects. The impact of CFS on various aspects of L. monocytogenes, such as biofilm formation, auto-aggregation, cytotoxicity, hemolysin activity, adhesion, and invasion, was also investigated. CFS treatment effectively inhibited biofilm formation at concentrations ranging from 1/16MIC (9.67%) to MIC (80%). It also reduced preexisting biofilms by 4.2%–70.91%. The CFS demonstrated cytotoxic effects on Caco-2 cancer cells (IC50, 44.64 mg/mL) and diminished hemolytic activity of L. monocytogenes at concentrations from MIC to 1/4 MIC, with reduction percentages of 50.66% and 28.66% respectively. Moreover, CFS treatment suppressed the expression of virulence genes, inhibiting the pathogenicity of L. monocytogenes. In conclusion, Lpb. plantarum TW57-4 exhibits favorable probiotic properties, antibacterial effects, and holds potential as a natural bacteriostatic agent. Further research and practical applications are warranted. It also shows promise as a bio-intervention for controlling L. monocytogenes biofilm in the food industry.

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