Bacteriocins have attracted much attention in the field of biopreservation and human therapeutics. Therefore a study was carried out to isolate a bacteriocin producing lactic acid bacteria from the fermented appam batter using MRS medium. The bacteriocin produced by the isolate was active against Listeria monocytogenes MTCC 657 and Acinetobacter baumannii MTCC 1425 among the test organisms examined. The bacteriocin producing organism was identified as Pediococcus pentosaceus by standard microbiological methods and 16s rRNA sequencing. Media optimization was carried out by altering the initial pH, time and temperature employed in the production of bacteriocin. In this study, enhanced bacteriocin production was observed at pH 6.0, temp 30 °C and 24 h. The concentration of major carbon and nitrogen sources were also studied for their influence on bacteriocin production.  It was found that the bacteriocin activity was increased when the carbon source –dextrose concentration was increased in MRS medium. However, nitrogen source did not have significant impact on bacteriocin production. This indicates the organism P. pentosaceus produced bacteriocin and exhibited significant growth inhibition against the indicator organisms. Therefore, the bacteriocin can be either used as a biopreservative or as an antibiotic to treat clinical pathogens.