Millets are grains that have high nutritive values. The current study aimed at studying the effect of prebiotics on the production of bacteriocins and their antibacterial activity. Bacteriocins were extracted from probiotic species of Lactobacillus plantarum and Lactocaseibacillus rhamnosus grown in millet modified medium and MRS medium. Results of anti-microbial assays of bacteriocin indicate enhanced activity in presence of proso millet. Bacteriocin isolated from L. rhamnosus growing in proso modified media had a zone of inhibition 15 ± 0.35mm against B. cereus and 16.8 ± 0.19mm against E. cloacae. Molecular weight was determined by SDS-PAGE and was found to be ~ 48 kDa. Thus, it could be concluded that proso millet acts as an efficient prebiotic. Crude, unprocessed extracts of millets were used in this study and significant results were obtained, suggesting that including them in everyday diet could render a source of natural prebiotic that is affordable and available commonly across the globe.