Foodborne pathogens such as Listeria monocytogenes and Staphylococcus aureus can persist in food processing environments by forming resilient biofilms that resist standard sanitation methods and serve as long-term sources of contamination. Biofilm growth is often controlled by quorum sensing (QS), including the autoinducer-2 (AI-2) system, which orchestrates communication within and between bacterial species and promotes group behaviors. Targeting QS provides a promising, non-lethal approach for controlling biofilms. Lactic acid bacteria (LAB), commonly used in foods, are known for improving safety and quality, and recent evidence shows their metabolites can interfere with QS and prevent biofilm formation without harming free-floating bacteria, thus lowering the risk of resistance. In this study, we tested a sterile, pH-neutralized (pH 6.5) cell-free supernatant (CFS) from a Pediococcus acidilactici cheese isolate for its ability to prevent mixed-culture biofilm formation by three L. monocytogenes strains from poultry on stainless steel surfaces. This LAB strain was selected from a larger collection because of its ability to inhibit AI-2 signaling in a Vibrio harveyi bioluminescence reporter assay. During biofilm formation, the CFS (prepared in MRS broth) was added at 50% (v/v) to Brain Heart Infusion (BHI) broth and incubated at 37 °C for 48 h under nutrient-matched conditions with and without CFS. Treatment with P. acidilactici CFS reduced L. monocytogenes biofilm cell counts on stainless steel by approximately 99% (2 logs), while leaving planktonic bacterial populations unaffected. The antibiofilm effect remained stable after heat treatment (100 °C, 5 min), indicating the presence of heat-resistant metabolites. Ongoing work aims to identify these QS-interfering compounds and further examine their effects on AI-2 signaling and biofilm development. Our main goal is to develop environmentally safe, QS-based biocontrol methods that could preserve beneficial microbiota while controlling harmful biofilms in food environments.