The Lactic Acid Bacteria (LAB) was isolated from yoghurt and identified as Lactobacillus delbrueckii subsp bulgaricus by biochemical methods and by 16s rDNA sequencing. The bacteria was screened for bacteriocin production by Agar well diffusion assay against four test organisms: Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis and Staphylococcus aureus, it was found to inhibit the growth of three test organisms except Klebsiella pneumoniae. The production of bacteriocin was optimized using Response Surface Methodology (RSM) based on the results obtained from single factor method. The optimized parameters were statistically validated using the software MATLAB version 188.8.131.522 (R 2007b) from Math lab works Inc. and assumed that Starch (3%), Casein (3%), FeSO4 (0.3%) and Tween 20 (0.24%) w/v supplemented to MRS media were optimum. Bacteriocin concentration before and after optimization was estimated by Lowry's method and was found to be 178.8μg/ ml and 310μg/ml respectively. © All Rights Reserved.