In this study, the probiotic potential of lactic acid bacteria isolated from rainbow trout intestine with inhibitory activity against Streptococcus iniae was evaluated by in vitro experiments. For this purpose, gram-positive and catalase negative lactic acid bacteria were screened based on probiotic characteristics such as acid and bile salt tolerance, antimicrobial activities against S. iniae and bio-safety assay including haemolytic activity and antibiotic susceptibility to eight major commercial antibiotics. At last, genotypic identification of candidate lactic acid bacteria isolates carried out by 16S rRNA gene sequence analysis. Based on results, among 148 bacterial isolates, five lactic acid bacteria strains belonged to the Lactococcus lactis subsp. were tolerant to pH=3-7.5, fish bile extract (10%) and had antimicrobial activity against S. iniae. Also, the bio-safety assay results revealed that none of the bacterial strains showed beta-haemolytic activity and all candidate bacterial strains exhibited phenotypic resistance to tetracycline. The multiple antibiotic resistant (tetracycline and chloramphenicol) was observed only in L. lactis NABRII61. This study showed that the five lactic acid bacteria isolated from rainbow trout intestinal microflora could be considered as probiotic strains in aquaculture. However, further experiments are required to confirm the nature of antibiotic resistance.