Columnaris is one of the oldest known fish diseases caused by Flavobacterium columnare. The wide spreading of the causative agent in freshwater environments and the susceptibility of fish to be attacked by it after mechanical and/or environmental factors makes F. columnare among the most prevalent pathogens in cultured, ornamental, and wild fish populations. One hundred fishes of tilapia (Oreochromis niloticus) were collected randomly from ponds of Central Laboratory for Aquaculture Research of Abbassa, Abou Hammad, Sharkia, Egypt for Flavobacterium columnare isolation. F. columnare was isolated from (150) out of the (350) examined samples. The bacteriological examination revealed that the prevalence of F. columnare was (42.8%) in Oreochromis niloticus. The highest prevalence of F columnare was in gills (33.3%) followed by skin (30%) then fins (24.6%) and kidney (12%). These isolates were further characterized by PCR that revealed positive result for 16S RNA gene with specific amplicons size 675bp. The result of antimicrobial sensitivity testing of the isolates revealed that doxycycline, erythromycin, tetracycline, trimethoprim, nalidixic acid and streptomycin were the most effective drugs against F.columnare.