Bacteria were studied for potential probiotic activity against vibriosis, yersiniosis and lactococcosis in rainbow trout. A total of 79 bacterial strains were isolated from rainbow trout rearing water, and screened for antagonistic activity against Vibrio anguillarum, Yersinia ruckeri and Lactococcus garvieae using a well-diffusion agar assay. Vibrio spp. showed inhibitory activity against V. anguilarum and L. garvieae, while Aeromonas spp. displayed antagonistic activity against L. garvieae. Antagonistic L. garvieae strains displayed inhibitory activity against all pathogens. Antagonistic strains were characterized for enzymatic activity (protease, lipase) and hydrophobicity. Vibrio sp. A12, and Aeromonas sp. A5, G1, were found to have enzymatic properties and hydrophobicity. L. garvieae strains showed weak hydrophobicity and did not display enzymatic activity. Phenotypic characteristics of antagonistic strains were determined by conventional API 20NE and API STREP rapid identification systems. Antagonistic L. garvieae strains were confirmed as L. garvieae by PCR using species-specific primers. Candidate probiotic strains were tested for pathogenicity in rainbow trout by intraperitoneal injection. Following a challenge, L. garvieae strains caused mortality and were eliminated from further study. As a result, Aeromonas spp. and Vibrio spp. were identified as probiotic candidates with the potential to control vibriosis and lactococcosis.