Kefir is a probiotic dairy product containing multiple species of lactic acid bacteria, acetic acid bacteria, and yeast, with varying microbial composition depending on geographical origin. In the present study, we characterized the acetic acid bacterial population in Korean kefir by next-generation sequencing-based community analysis and isolated a novel acetic acid bacterial strain, Acetobacter fabarum DH1801. To evaluate its potential application in the food industry, the antimicrobial activity of A. fabarum DH1801 against seven foodborne pathogens (Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii, Salmonella Enteritidis, enterotoxigenic Escherichia coli, and Shigella flexneri) was analyzed by growth curve analysis. Remarkably, the culture filtrate of the novel isolate inhibited the growth of all seven pathogenic bacteria in a dose-dependent manner, which was superior to acetic acid solution of same pH value. Our findings suggest that the A. fabarum DH1801 strain forms a protective barrier during kefir fermentation against contamination by foodborne pathogens.