Fish are susceptible to infectious diseases brought on by a variety of phylogenetically distinct bacterial pathogens. This study was aimed at the isolation and identification of Enterococcus faecalis in cultured catfish (Clarias gariepinus) and the detection of its effect on fish culture. For this purpose, 200 moribund and freshly dead Clarias gariepinus were randomly collected from private fish farms in the Ismailia governorate. The clinical signs, postmortem lesions, isolation and identification of Enterococcus faecalis as well as molecular identification were investigated. The clinical picture displayed hemorrhages, ulcers, macerated muscles on the flank, redness at the base of fins, enlarged liver, an engorged spleen, a congested kidney, and an inflamed heart. Results of bacterial investigation revealed (53%) of Enterococcus faecalis. The highest prevalence was in summer (39.6%), while the lowest was in winter (11.32).  The results of molecular identification revealed that all isolates harbored the EF3314 gene of Enterococcus faecalis at a fragment size of 566 bp. Finally, it could be concluded that Enterococcus faecalis is a threatening bacterial infection in Clarias gariepinus culture and it can be easily detected by molecular identification of the causative pathogen