Flavobacterium columnare is an important pathogen of freshwater fishes often causing high mortality. Seven strains of F. columnare have been isolated from gill necrosis, skin lesions and internal organs of Catla catla, Labeo rohita, Cirrhinus mrigala, Carassius auratus, Anabas testudineus and Clarias batrachus and characterized by biochemical reactions and serological tests viz indirect enzyme‐linked immunosorbent assay (ELISA), Dot‐ELISA, agglutination test. All the strains showed binding to Congo red dye as well as hemolysis. All the seven strains were susceptible to amikacin, gentamycin and ofloxacin. In vivo LD50 dose of virulent strain MS2 was found to be 6 × 104 CFU/mL after experimental infection to L. rohita. Serologically all the seven strains showed a positive result to agglutination, Dot‐ELISA and indirect ELISA. The agglutination titer was found to be in the range of 256‐131,072. The lysozyme activity of hyperimmune sera was found to be 39.37 ± 0.80 units/mL. This is the first extensive study that report about the various strains of F. columnare from freshwater fishes of Eastern India and their differentiation by biochemical and serological methods.