One of the major threats to aquaculture sector is bacterial diseases. In the current study, samples from different species of cultured fishes were collected from different fish farms at different localities in Menofiya and Kafr El-Sheikh Provinces, Egypt for the detection of causative agents of some disease. Clinically, the infected fishes were inspected for symptoms like darkness of skin and, hemorrhages in different parts of the fish's body including the base of fins, tail, and rot of fins. Identification of the retrieved bacterial pathogens using conventional biochemical and molecular methods were demonstrated. Based on the phenotypic and biochemical characterization using API20E, API 20 NE and VITEK® 2 compact, the retrieved isolates were identified as Yersinia ruckeri and Psychrobacter species. Molecular characterization was accomplished through polymerase chain reaction (PCR) and confirmed by DNA-sequencing and phylogenetic analysis. The high molecular identity and close phylogenetic relationship confirmed that the isolates were genetically homologous to Yersinia ruckeri and Psychrobacter spp.