A serious infectious disease of unknown aetiology recently broke out climbing perch (Anabas testudineus) farms in Vietnam, with moderate to high mortality rates during outbreaks. Natural infection was characterized by darkened body colour, eyes with corneal opacity, listless swimming, ascites, hepatomegaly, and splenomegaly. Pure small and opaque colonies were observed and Gram-positive cocci, catalase negative and oxidase negative, were isolated in brain heart infusion (BHI) agar and blood agar (BA) from internal organs of diseased fish. Therefore, the objectives of this study were aimed at identifying bacterial causative agents isolated from diseased climbing perch, evaluating the pathogenicity of the pathogen in climbing perch, determining the susceptibility of the pathogen isolates to anti-microbial agents, and analysing histopathological changes in infected climbing perch. In this study, conventional and rapid identification systems, and 16S rRNA gene partial sequencing were used to identify the causative agents of the disease. Streptococcus iniae made up 69% of the total streptococcal species identified 26% was S. agalactiae and Gemella hydrolysans remained less than 5%. Susceptibility of the S. iniae and S. agalactiae isolates to 11 antibiotics was tested using the disc diffusion method. LD₅₀ trial performance of S. iniae and S. agalactiae showed the virulence of these isolates in climbing perch and fulfilled Koch's postulates. Most of infected fish in the experiments showed clinical signs similar to the natural infection. Histopathological lesions of internal organs of climbing perch infected with streptococcosis showed that hemolysis at the kidney and granuloma-like lession at the spleen were observed. To our knowledge, this is the first report of S. iniae and S. agalactiae as pathogens of climbing perch.