To assess the biofilm-forming ability of Listeria monocytogenes and its susceptibility to food-grade biocides, food (n = 120) and food contact equipment samples (n = 60) collected from the ready-to-eat meat-based food chain were analyzed. A total of 113 L. monocytogenes isolates were obtained and genetically characterized. Nineteen strains were tested for biofilm-forming ability and susceptibility to benzalkonium chloride, sodium hypochlorite and nisin. Most strains were moderate to strong biofilm-formers (crystal violet optical density ranging from 0.13 ± 0.03 to 0.20 ± 0.04). When treated with benzalkonium chloride and sodium hypochlorite, most biofilms were reduced, but the same did not happen with nisin. Three strains revealed a resistant profile to all biocides with high estimated LD90 values (≥1.79 mg/ml for benzalkonium chloride and sodium hypochloride and >1000 IU/ml for nisin). Biofilm-forming ability and LD90 values highlight the need to consider other sanitizers and novel strategies for mitigation and control of L. monocytogenes biofilms.