Biofilms that contain foodborne pathogens and form on food contact surfaces pose a persistent challenge to food safety and public health. This study evaluated the biofilm inhibitory potential of sterile and neutralized cell-free supernatants (CFSs) derived from two foodborne lactic acid bacteria (LAB) strains (Enterococcus faecium LFMH-B79b and Pediococcus acidilactici LFMH_B10) against both single- and mixed-species biofilms of four target bacterial species that negatively affect food quality: Pseudomonas fluorescens, Staphylococcus aureus, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes. Bacteria in saline suspensions (8 log CFU/mL) of the target species were allowed to attach for three hours at 20 °C on model stainless steel surfaces, under either single- or mixed-species conditions, and then incubated in tryptic soy broth (TSB), with or without the addition of each LAB CFS (at 50% v/v), for up to 48 h at 20 °C. At 24 and 48 h of incubation, both planktonic and biofilm bacteria on surfaces were counted using selective plate counting. Our results indicated that in single-species biofilms, S. Typhimurium sessile cell counts were reduced by approximately 90% (1 log) after 48 h of incubation with the CFS of the E. faecium strain. A similar trend was observed in mixed-species biofilms, where co-incubation with that CFS again led to a ~1 log reduction in S. Typhimurium biofilm cell numbers. It should be noted that the observed inhibition of biofilm formation was not due to the inhibition of planktonic growth or nutrient dilution, indicating a targeted inhibitory effect of E. faecium-derived metabolites on S. Typhimurium biofilm formation. No significant biofilm inhibition was observed for the other three target bacterial species. These findings provide a strong rationale for further exploring LAB-derived metabolites for biofilm-targeted interventions in food systems, highlighting the complex nature of intercellular microbial interactions.