Bacteriocins are secreted proteins produced by lactic acid bacteria (LAB) in early stationary phase of growth, which can inhibit the growth of similar or closely related bacteria. In this study, we attempted to screen Bifidobacterium isolate that produced bacteriocin exhibiting antibacterial activity against Clostridium difficile. Among 800 LAB strains isolated from calf fecal samples, 620 strains (77.5%) exhibited antimicrobial activity against C. difficile, of which 21/620 were identified as Bifidobacterium sp. Neutralized cell-free supernatant (NCFS) of the selected Bifidobacterium isolate, BD-1, showed an antimicrobial effect against not only drug resistant C. difficile (strain 630) but also Bacillus subtilis, Pediococcus pentosaceus and E. coli ETEC. The maximum growth observed by optical density was reached approximately 5.8 after 18 h of incubation. Furthermore, the production of antimicrobial compound was found at the late exponential phase of growth (12h) and reached a maximum activity during the stationary phase. The anti-bacterial property of the NCFS fraction of the BD-1 strain was heat (100ºC for 30 min) and pH-stable (pH 3-9); however, it was sensitive to protease treatment. Our findings suggested that the substance(s) in NCFS of Bifidobacterium isolate BD-1 could potentially be a bacteriocin and may be a good candidate for alternative antimicrobial agents to treat C. difficile infection.