LuxS/AI-2 quorum sensing is involved in the virulence of many bacterial pathogens, including the fish pathogen Edwardsiella tarda. In a previous study, we identified a small peptide, 5906, which inhibits E. tarda LuxS activity by binding specifically to LuxS in a manner that probably prevents the formation of functional LuxS homodimer. In the present study, using Japanese flounder as the experimental animal, we analyzed the antibacterial effect of 5906 produced by DH5α/p5906 (an Escherichia coli strain that produces 5906) and pID5906 (a mammalian plasmid that functional in flounder constitutively expresses 5906) against different bacterial fish pathogens. We found that fish administered with both DH5α/p5906 and pID5906 exhibited reduced bacterial recovery following E. tarda challenge. We also examined the effect of 5906 on the infection caused by another two fish pathogen, Aeromonas hydrophila and Vibrio harveyi. The results indicated that 5906 produced by DH5α/p5906 inhibited the AI-2 activity of A. hydrophila and V. harveyi, and that fish administered with DH5α/p5906 showed enhanced resistance against challenges with both bacteria. These results suggest that 5906 or its analogues/derivatives may be exploited for the development of broad-spectrum antibacterial agents applied in the prevention and control of fish bacterial diseases.