Vibrio anguillarum and Vibrio harveyi are two important pathogens that affect a wide range of fish species. In this study, we developed a formalin-inactivated whole-cell bivalent vaccine (named IVVah1) against V. anguillarum and V. harveyi and examined its effect in a turbot model. The results showed that when administered via a single intraperitoneal (i.p.) injection, the vaccine induced high rates of relative percentage survival (RPS) against V. anguillarum and V. harveyi from the third week post-vaccination (wpv). The RPS reached 94.74% and 91.89% against V. anguillarum and V. harveyi, respectively, at 4 wpv, and remained high (>80%) till at least 8 wpv. Specific antibodies were produced in the vaccinated fish at 1 wpv and peaked at 2 to 3 wpv. Secondary antigen exposure induced strong proliferation of peripheral blood leukocytes (PBLs) and increased the number of monocytes in PBLs. Compared to the control fish, the vaccinated fish exhibited normal growth rates and were able to thoroughly clear the challenging bacterial pathogens from tissues. These findings indicate that IVVah1 is a highly protective bivalent vaccine with a good potential to be used in aquaculture against V. anguillarum and V. harveyi infection.