Bacteriocins are ribosomally synthesized antimicrobial peptides produced by lactic acid bacteria. The objective of this study is to investigate antibacterial and antifungal spectrum of bacteriocin that was purified and characterized from Lactobacillus plantarum MTCC 9496 and to determine toxicity of this peptide, named plantaricin 9496 on eukaryotic cells. Plantaricin 9496 was purified by adsorption desorption method followed by gel permeation chromatography. Molecular weight of plantaricin 9496 was found to be of 5.8 kDa. It exhibited broad antimicrobial spectrum against fruit and vegetable spoiling organisms, especially Staphylococcus aureus, Listeria monocytogenes, Pseudomonas fluorescens, Bacillus subtilis, Aspergillus niger, Fusarium oxysporum, Botrytis cinera, Penicillium expansum, Rhizopus stolonifer and Candida albicans. Plantaricin 9496 retained activity after heating upto 100°C and at pH range of 2.0-7.0. Cytotoxicity of plantaricin 9496 on spleenocytes showed that it was non toxic upto concentration of 40- 2600 µg/ml and CC50 value is above 20800 µg/ml, indicating that cytotoxicity of plantaricin 9496 is very low. Hence, this bacteriocin can be used as safe biopreservative to extend shelf life of perishable food products.