This study sought to confirm the inhibitory ability of Clostridioides difficile KCTC 5009 strain using the bacteriocin-producing Lactococcus lactis CJNU 3001 strain. For this purpose, bacterial culture and partially purified bacteriocin verified the antibacterial activity against C. difficile strain. As a result, both samples showed strong antibacterial activity against the target strain. Co-culture tests were performed to more closely analyze the competition between the two strains. As a result, a similar inhibitory effect was confirmed in RCM broth and CMM (colon model medium) broth. In particular, inhibition of C. difficile KCTC 5009 strain was not observed until the exponential phase of the growth of L. lactis CJNU 3001 strain, because bacteriocins were produced during that period. It was inferred that it actively suppressed the growth of the target strain after that. Therefore, as it was proven that the bacteriocin-producing L. lactis CJNU 3001 strain can effectively control C. difficile strain in co-cultures, it was judged that it could be an alternative for CDI (Clostridioides difficile infection) treatment.