After enrichment of Odontesthes platensis intestinal contents, 53 lactic acid bacteria (LAB) were isolated. From the four isolates that showed inhibitory activity against Lactococcus garvieae 03/8460, strain TW34 was selected because it exerted the strongest inhibition. It also inhibited other Gram-positive bacteria, but not Gram-negative fish pathogens. Phenotypic and 16S rDNA phylogenetic analyses showed that TW34 belongs to Lactococcus lactis. In addition, TW34 showed to be sensitive to different antibiotics. The production of the inhibitory agent against L. garvieae was growth associated, and it was significantly influenced by the incubation temperature. The optimal temperature for the antimicrobial production was as low as 15°C. Both acidification and hydrogen peroxide production were ruled out as the source of inhibition. In contrast, the antimicrobial activity was completely lost by treatment with proteolytic enzymes, which confirmed that the inhibitory substance was a bacteriocin. The bacteriocin was highly thermostable (121°C for 15 min) and active between pH 3 and 11. It remained stable for up to 2 months when stored at 4°C and up to 6 months at −20°C. Our results suggest that the strain L. lactis TW34 could provide an alternative for lactococcosis control and therefore be considered for future challenge experiments with fish.