Vibrio vulnificus can cause infections in aquaculture-raised fish and is considered an opportunistic human pathogen. We isolated V. vulnificus from diseased hybrid tilapia (Oreochromis niloticus X O. aureus) cultured in a North American water reuse aquaculture facility. We have characterized the isolate using biochemical and molecular methods, developed a disease infection model, and determined that formalin-inactivated whole-cell vaccine provides protection against V. vulnificus. The V. vulnificus isolate was determined to be biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2. Fish vaccinated with the formalin-inactivated whole-cell vaccine responded to vaccination as measured by agglutinating antibody titer. In two separate trials, vaccinated tilapia exhibited relative percent survival of 73 and 60% following challenge with the homologous isolate. In additional trials, vaccinated tilapia exhibited survival values of up to 87.5% following challenge with a heterologous isolate. Use of a mineral oil adjuvant enhanced protection.