The antimicrobial effects of ten isothiocyanates (ITCs) present in cruciferous vegetables and radish root hydrolysate were investigated against pathogenic bacteria from olive flounder. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were measured against two gram-positive bacterial strains (Streptococcus parauberis, S. iniae) and four gram-negative bacterial strains (Edwardsiella tarda, Vibrio ichthyoenteri, V. harveyi, Photobacterium damselae) by using a broth microdilution technique. The antibacterial activity of ITCs was in the order sulforaphane > sulforaphene > phenylethyl ITC > erucin > benzyl ITC > iberin > I3C > allyl ITC > phenyl ITC > hexyl ITC. The susceptibility of fish pathogens to ITCs was in the order of V. harveyi > E. tarda > P. damselae > S. parauberis > S. iniae > V. ichthyoenteri. Antimicrobial activity (MIC) of radish root hydrolysate was 0.250 mg/mL against S. iniae, 0.438 mg/mL against S. parauberis, and 0.500 mg/mL against both E. tarda and V. harveyi. The aliphatic ITCs were potent inhibitors of the growth of fish pathogens, followed by aromatic ITCs and indolyl ITC. The presence of a double bond in the chemical structure of ITCs decreased antibacterial activity, while ITCs with a thiol (-S-) group and a longer carbon chain increased antibacterial activity. These results suggest that ITCs have strong antibacterial activities and may be useful in the prevention of fish pathogens.