This study reports the production and characterization of a novel antibacterial polypeptide, designated laparaxin, which is secreted by Lactobacillus paracasei NRRL B-50314. Crude laparaxin has antibacterial activity against a wide variety of Gram-positive bacteria, including: lactic acid bacteria (Lactococcus lactis and Lactobacillus buchneri), food-borne pathogens (Listeria monocytogenes), gastrointestinal pathogens (Enterococcus faecalis), and opportunistic pathogens (Staphylococcus aureus methicillin-sensitive (MSSA) and methicillin resistant (MRSA) strains, a hetero-vancomycin-intermediate methicillin resistant strain (Hetero VISA also MRSA) MM66, and homogeneous vancomycin intermediate (Homo VISA). Using L. lactis as an indicator strain, the inhibitory activity of crude laparaxin was detected originally in early log phase, and the activity maximizes at the early stationary phase and remains stable after prolonged incubation. Laparaxin activity is stable after 30 min of incubation at 94°C. Higher concentrations of inhibitory activity are produced when glucose, fructose and sucrose are used as carbon-sources in growth media. Crude laparaxin has potential applications in food and feed industries, as well as in clinical and veterinary medicine.