The olive flounder (Paralichthys olivaceus) is an important mariculture fish in Korea, but its farming has been negatively impacted by bacterial diseases such as streptococcosis. Here, we sought new methods for identifying and characterizing the S. iniae and L. garvieae populations present in fish farms located along the southern coast of Korea. Consistent with the type strains S. iniae ATCC29178 and L. garvieae KG9408, the bacterial isolates from olive flounder were all found to be catalase and oxidase test-negative and non-motile. Although the bacterial isolates were phenotypically similar, differences were noted in the results of the Voges-Proskauer (VP), alkaline phosphatase (PAL) and hemolysis tests. PCR using specific primers was used to confirm that the isolates were S. iniae or L. garvieae, and SDS-PAGE and immunoblot assays were used to compare the cell lysate and antigenic protein profiles of each isolate with that of the appropriate type strain. The results of the biochemical tests and PCR were highly consistent with the findings of our SDS-PAGE and immunoblot assay, indicating that the SDS-PAGE and immunoblot protocols could be used for identification and tentative classification of the causative agents of olive flounder streptococcosis.