Antimicrobial resistance in aquaculture pathogenic bacteria is a prevalent threat that contributes to the increasingly uncontrolled misuse of antibiotics. Currently, bacteriocins have been selected as a viable alternative to antibiotics in aquaculture due to their high safety profile. In this study, Colicin N from Escherichia coli was cloned, expressed, and purified. Among seven tested Vibrio strains, recombinant Colicin at a concentration of 25 µg/mL exhibited inhibitory activity against Vibrio parahaemolyticus and Vibrio owensii. Furthermore, recombinant Colicin N showed a significant inhibitory effect against Vibrio vulnificus and Vibrio harveyi at a concentration of 12.5 µg/mL. The results of this study demonstrate the in vitro inhibitory activity of Colicin N against several pathogenic Vibrio species, highlighting its potential for further development as an antimicrobial agent in shrimp aquaculture.