The focus of the present study was to characterize chimeric synthetic plantaricin F which naturally produced by Lactobacillus plantarum against zoonotic pathogenic bacteria Staphylococcus aureus and Escherichia coli as antibacterial peptide. The syntheticbacteriocin by bioinformatics revealed higher stability under studied parameter, hence was taken up for further investigation. The amino acids of bacteriocin from L. plantarum were analyzed by SnapGene. Further, synthetic PLNF was characterized in silico. The translated partial amino acid sequence of the synthetic PLNF gene displayed 253 amino acids for whole and 148 without tag. The predicted properties of the peptide included theoretical isoelectric point (pI) and hydrophobicity was highly acidic. Molecular weight was 27.2KDa for whole protein and 15.8 KDa for without tag. Predication the molecular approach of using SnapGene software and the protein was having antingcity against bacteria and has B-cell epitope on the surface of protein. Prediction data base on characterization of bacteriocin is novel and predicts synthetic PLNF to be a peptide responsible for antimicrobial activity. The study provides information about a broad spectrum bacteriocin in native probiotic culture and paves a way towards its application as alternative natural antimicrobial agent against zoonotic pathogenic bacteria. Finally, the 3D peptide structure analysis in present study showed that the predicted structure of model and has more functional properties and probably the form most suitable for binding to bacterial cell walls.