The potential of transferring coliform bacteria present in cow manure applied to agricultural soil to fruits, vegetables and water is of great concern to farmers. In this study, we investigated the effect of supplementing cow manure with bokashi on survival of coliform bacteria during fermentation or pretreatment. The pretreatment of cow manure (4.5% total solids) supplemented with 5 - 20 g dry bokashi in sterile 250-ml Wheaton glass serum bottles (working volume, 100 ml) was conducted aerobically or anaerobically at 35 and 120 rpm in a shaker incubator for 48 h. Using microbiology and molecular techniques, samples (5 ml) were drawn at 12-h intervals and analyzed for fecal coliforms and probiotic microorganisms during the pretreatment. Our findings show that bokashi completely killed coliforms in cow manure within 24 h in both anaerobic and aerobic pretreatment conditions. Additionally, 16S rRNA gene analysis of bacteria present in bokashi showed the presence of Bacillus and Paenibacillus species, whereas that of cow manure pretreated with bokashi revealed the presence of manure derived probiotics such as Lactobacillus, Bacillus and Paenibacillus species. Further, heat-sterilized bokashi also effectively killed coliforms during cow manure pretreatment, suggesting that the ability of bokashi to kill coliforms may be attributed to the prebiotic (complex carbohydrates) characteristic of bokashi and not its probiotic content. Collectively, bokashi serves as prebiotic for rapid proliferation of manure-derived probiotics, eradication of coliforms in pretreated cow manure, and potentially help to prevent the spread of pathogens in the environment.