Technical Abstract: Streptococcus iniae, a Gram-positive bacterium, and Vibrio vulnificus, a halophilic Gram-negative bacterium, have been associated with severe disease impacting tilapia aquaculture. Recent reports suggest both bacteria have been associated independently and concomitantly with disease on commercial farms. Monovalent vaccines have been developed for disease control; however, the most effective delivery strategy has been via intraperitoneal (IP) injection. Due to handling stress and the cost associated with injecting each fish, a better strategy is to combine the monovalent vaccines into bivalent formulations. The objective of the present study was to test the ability of a killed bivalent S. iniae and V. vulnificus vaccine delivered by IP injection at protecting sex reversed hybrid tilapia (Oreochromis niloticus X O. aureus) against challenge with each bacterium, independently. In two independent trials, vaccination of tilapia with the bivalent vaccine conferred protective immunity against V. vulnificus and S. iniae as demonstrated by significant differences (P < 0.05) in survival curves between the sham-vaccinated and vaccinated groups. Relative percent survival values ranged from 79 to 89% for V. vulnificus and 69 to 100% for S. iniae following challenge of bivalent vaccinated fish. Use of this bivalent formulation may be a cost-effective strategy to reduce losses in tilapia co-infected with these two important bacterial pathogens.