Bacterial septicemia are responsible for severe losses in fish farms over the world. This study aimed to investigate the Enterobacteriaceae infections in sharptooth catfish Clarias gariepinus. A total of 120 catfish were collected from small canal in Farshout, Qena Governorate, and were subjected to clinical and bacteriological examination. Catfish sampled showed signs of typical hemorrhagic septicemia on the skin and internal organs. Bacteriological investigations resulted in isolation of 240 isolates that belong to Enterobacteriaceae. Interestingly, conventional biochemical identification showed that 16 isolates are suspected to be Citrobacter spp. Based on genetic sequence analysis of the 16S rDNA, only 8 (50%) of the suspected isolates were identified as Citrobacter freundii. To investigate the pathogenicity of isolated C. freundii strains to catfish, an experimental challenge experiment was conducted where challenged fish showed signs and lesions of acute septicemia. The current study is the first to report Citrobacter infections in catfish in Upper Egypt.