A total of 199 microorganisms were isolated from the intestinal contents of flounder (Paralichthys olivaceus) in a fish farm in Seoul, Korea. Among these strains, DS-12 was selected as a candidate for flounder probiotics because of its excellent exhibition of antimicrobial activity against fish pathogens such as edwardsiella, pasteurella, aeromonas, and vibrio, and initiate growth in 10% NaCl, 10% bile, and in broth at pH 3 for 90 min. This strain was Gram-positive, and catalase-negative coccoid rods that produced gas from glucose and formed more than 90% of lactate as the D(−) isomer. This organism is positioned at a cluster in the genus Weissella on the phylogenetic tree based on 16S rRNA sequences, which were assigned to Weissella hellenica on the basis of DNA-DNA relatedness. However, the type strain of W. hellenica JCM 10103^T had no antibacterial activity against the fish pathogenic bacteria and was found to be quite different from the DS-12 strain in some sugar fermentation patterns of α-methyl-D-glucoside, esculine, cellobiose, melibiose, D-raffinose, and D-turanose, being especially unable to grow at 15 and 35°C in 7% NaCl and 10% bile. The results obtained in the present study demonstrated that the type strain of W. hellenica had no probiotic characteristics, but the strain DS-12 could be used as a specific probiotic for flounder.