A study was conducted to evaluate the probiotic properties of endogenous rainbow trout microbiota against pathogenic Lactococcus garvieae. A total of 335 bacterial strains were isolated from rainbow trout and screened for antagonistic activity against L. garvieae using an agar spot assay. Antagonistic strains were grouped by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S rRNA gene sequence analysis. The results revealed that the antagonistic strains belonged to the genera Lactobacillus, Lactococcus and Leuconostoc. Further probiotic characteristics, such as specific growth rate, doubling time, resistance to biological barriers, antibiotic resistance, hydrophobicity and production of antimicrobial substances, were also studied. These strains were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The antagonistic efficacy was maintained after sterile filtration and was sensitive to proteinase K, indicating that proteinaceous extracellular inhibitory compounds were at least partially responsible for pathogen antagonism. Based on these results, these strains should be further studied to explore their probiotic effects in challenge experiments in vivo. This study shows clear evidence that the indigenous trout-associated microbiota may provide a defensive barrier against L. garvieae.