Bacteriocins are the most important natural preservatives against pathogens, but their limited spectrum could not fully ensure food safety. In this study, bacteriocin producers showing specific-antimicrobial activity to foodbome pathogens were isolated and the success of the cocktail of pathogen-specific bacteriocins (PSBs) in milk were demonstrated. PSB producers were isolated from 250 different foods by a new approach that screening their antimicrobial activity against a mixture of five different strains of each Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus. Lactobacillus plantarum PFC339, Enterococcus faecalis PFC340, Lactobacillus delbrueckii subsp. lactis PFC341 were identified that inhibited the growth of pathogen strains respectively, but not lactic acid bacterial strains. The molecular sizes of PSBs were 1219.021, 3346.803, 4853.768 Da respectively. They inhibited by disrupting the permeability or creating pores on the cell walls. While 100 AU/mL decreased the amount of pathogens in milk which they are specifically active, each PSBs did not show any inhibitory effect on other two pathogens. However, PSBs cocktail was able to stop the growth of all inoculated pathogens. In conclusion, three bacteriocin producers specifically inhibiting B. terms, L. monocytogenes, S. aureus were isolated and the growth of pathogens in food systems were prevented by use of these PSBs as cocktails for the first time.