Lactic acid bacteria (LAB) are able to secrete antimicrobial peptides called bacteriocins, which inhibit other bacteria such as pathogenic microorganisms. Therefore, bacteriocin-producing starters can be used as natural biopreservatives for various foods. The objective of this study was to screen and characterize bacteriocin-producing LAB from Kimchi and to investigate their applicability as a starter in Kimchi fermentation. To screen bacteriocin-producing LAB, gram-positive and gram-negative bacteria were used as indicators. To measure the antimicrobial activities of isolates, agar well diffusion assay method was used. According to the results, bacteriocin produced by Lb. sakei B16 showed antimicrobial activity against Listeria monocytogenes ATCC 19115, Escherichia coli KCTC 1467, and Lactobacillus plantarum KTCT 3104. Furthermore, bacteriocin was very stable after treatment with high temperature and high and low pH, but its effects were inhibited by treatment with proteolytic enzymes such as trypsin, proteinase K, and α-chymotrypsin, revealing their bacteriocin-like protein-based structure. These results suggest that Lb. sakei B16 and its bacteriocin are good candidates as a functional probiotic and natural biopreservative, respectively, in fermented foods.