Columnaris disease caused by Flavobacterium columnare exists worldwide and affects a wide variety of fishes. This study aimed to investigate the isolation and characterization of Flavobacterium columnare from naturally infected fishes (Oreochromis niloticus and Clarias gariepinus) in Ismailia, using conventional biochemical methods and molecular techniques. Bacteriological examination revealed presence of 162 suspected Cytophaga isolates of which only 102 isolates were characterized as F. columnare strains according to colony character on Cytophaga agar which appeared yellow or whitish yellow in color, flat irregular with stiletto edge, clear swarming and rhizoid characters. Biochemical characterizations demonstrated that all isolates tested uniformly positive for catalase, gelatin liquefaction, absorption of Congo Red and flexirubin pigment, but they were negative for indol, Methyl red, Vogus Proskauer, and lactose, glucose, saccharose, manitol, arabinose and maltose fermentation. All isolates produced unique and clear PCR bands of the expected size (675 base pair). Consequently, PCR confirmed the positive results of the biochemical reactions of suspected isolated strain and concluded that it is of  F.columnare.