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Natural antimicrobial peptides, produced by beneficial bacteria, are urgently needed to combat harmful microorganisms and fulfill consumer’s preference of clean label. However, the commercial implementation of natural antimicrobial peptides is hampered by the cost and the composition of the production medium. Therefore, finding low cost production media will facilitate their industrial application. The study described here investigated the production of antimicrobial peptides in dairy acid whey using novel food-grade lactic acid bacteria (LAB). Enterococcus durans OSY-EGY, isolated from artisanal cheeses, and E. faecalis OSY-RM6, isolated from raw cow’s milk, were found to successfully produce their enterocins EGY and RM6 respectively in the ferment acid whey. Additionally, to optimize enterocins EGY and RM6 production in the fermented whey, the effect of incubation time (24, 48 and 72 hours), growth temperature (30ºC and 37ºC), initial pH (4.3, 6.0, 6.5 and 7.0), the chemical composition of the whey (adding 1% yeast extract, 1% lactose or both) and the inoculation volume of the bacterial culture (1% and 2%) were explored. Finally, to facilitate further industrial application, the produced enterocins were semi purified and concentrated from the fermented whey using a new simple purification protocol using resin beads followed by ultrafiltration and freeze drying. The concentrations of EGY and RM6 in the resulted active antimicrobial powders as two final products were 512,000 AU/g and 128,000 AU/g respectively. To our knowledge, this is the first report of antimicrobial powder derived from fermented acid whey.