Aims: This study was designed to investigate the ability of naturally occurring bacteria isolated from mushroom substrate to prevent biofilm formation by Listeria monocytogenes or to remove existing biofilms in mushroom production facilities. Methods and Results: It is generally recognized that L. monocytogenes forms biofilms that can facilitate its survival in food-processing environments. Eleven bacteriocin-producing isolates were identified and the bacteriocins characterized based on heat and enzyme inactivation studies. Further characterization was undertaken by MALDI-TOF mass spectrometry, PCR and sequencing. Production of nisin Z (by Lactococcus lactis isolates), subtilomycin (by Bacillus subtilis isolates) and lichenicidin (by Bacillus licheniformis and Bacillus sonorensis isolates) was detected. In co-culture with L. monocytogenes, the bacteriocin-producing strains could prevent biofilm formation and reduce pre-formed biofilms. Conclusions: Mushroom substrate can be a source of bacteriocin-producing bacteria that can antagonize L. monocytogenes. Significance and Impact of the Study: The results highlight the potential of bacteriocin-producing strains from mushroom substrate to reduce L. monocytogenes biofilm in food production environments, contributing to a reduction in the risk of food contamination from the environment.