In Japan, mass mortalities of cultured marine fish due to bacterial diseases have been often reported. Among diseases caused by Gram-positive bacteria, Lactococcus garvieae infection in yellowtail, amberjack, jack mackerel and striped jack, Streptococcus iniae infection in yellowtail, Japanese flounder and filefish, S. parauberis infection in Japanese flounder, Nocardia seriolae infection in yellowtail. Among diseases caused by Gram-negative bacteria, Edwardsiella tarda infection in Japanese flounder and red sea bream, Photobacterium damsela subsp. piscicida infection in yellowtail, red sea bream, striped jack and Japanese flounder, Tenacibaculum maritimum infection in Japanese flounder, red sea bream and striped jack, Vibrio spp. infection in various marine fishes cause serious damages every year. A commercially available L. garvieae and V. anguillarum vaccine is used in yellowtail and amberjack by oral administration or injection. S. iniae vaccine is used in Japanese flounder by injection. Recently, oil-based bivalent vaccine against P. damsela and L. garvieae infection was developed in yellowtail and amberjack. We have developed pentavalent vaccine against streptococcicosis and edwardsiellosis in Japanese flounder. Efficacy of pentavalent vaccine with formalin-killed S. parauberis (serotype I and Ⅱ strain), S. iniae and E. tarda (motile and non-motile strain) were tested by vaccination followed by intramuscular challenge with the each pathogen. The Japanese flounder vaccined with pentavalent vaccine showed higher resistance against the each pathogen on two weeks post-vaccination. These results revealed that pentavalent vaccine was effective in prevention against streptococcicosis and edwardsiellosis in cultured Japanese flounder.