Preventing the introduction of Listeria monocytogenes, subsequent biofilm formation, and persistence in food processing environments is important for reducing the risk of cross-contamination of ready-to-eat foods. This study determined the effect of Ɛ- poly-lysine (EPL), hydrogen peroxide (HP), and lauric arginate (LAE) on L. monocytogenes biofilm formation and the inactivation of mature biofilms. For inhibition studies, biofilms of L. monocytogenes Scott A (serotype 4b) and 2014L-6025 (1/2b) were developed separately at 37°C for 48 h in the presence of sub-inhibitory concentrations (SIC) of either EPL (10 ppm), HP (2 ppm), or LAE (1.5 ppm) on polystyrene plates and stainless-steel rounds. Inactivation was determined by exposing mature biofilms on each surface to each antimicrobial at their minimum bactericidal concentration (MBC), 10xMBC, or 100xMBC for 24 h at 37°C. The presence of these antimicrobials at SIC did not inhibit biofilm formation on either surface and their effect on mature biofilms varied by strain and surface. Application of EPL at 1xMBC (100 ppm) for 24h resulted in greater reductions in counts of both strains on polystyrene than HP (40 ppm) and LAE (5 ppm) under the same conditions at 1xMBC (P ≤ 0.0243). Exposure of mature biofilms to LAE at 10xMBC (50 ppm) for 1h was more effective in reducing counts on polystyrene than HP at 10xMBC (400 ppm) for the same duration (P ≤ 0.0136), and both HP and LAE applied at 100xMBC (4000 and 500 ppm, respectively) for 24 h more effectively inactivated mature biofilms of L. monocytogenes Scott A on polystyrene compared to EPL (10,000 ppm) (P ≤ 0.0307). Application of LAE at 10xMBC for 24 h was more effective at inactivating strain Scott A on stainless steel compared to 10xMBC of EPL (1,000) or HP (P ≤ 0.0430). Future studies are needed to determine the efficacy of these and other antimicrobials on additional strains and serotypes of L. monocytogenes at temperatures relevant to food production and storage.