The Nile River in Egypt holds paramount importance as a natural freshwater fishery, capable of providing substantial fish yields. Among the pathogens affecting Nile tilapia (Oreochromis niloticus), Aeromonas hydrophila is the most agent causing high mortality. In the June 2023 to September 2023, around 120 moribund and freshly dead Nile tilapia were gathered from the Nile River, Al- Munib, Cairo, Egypt. The clinical symptoms in these fish were consistent with Motile Aeromonas Septicemia (MAS), including indications of surface hemorrhage, erosions, loss of scales, and some samples even showed exophthalmia, eye cataract and skin discoloration. Postmortem assessments indicated internal organ deterioration, enlargement of gall bladder, pale liver, yellowish fluid in the abdominal cavity and pigmentation on surface of the liver. Identification of A. hydrophila was accomplished through phenotypic characterization, diagnostics using SRO, GN24, features and homology of 16S rRNA gene sequences. Antibiogram analysis revealed sensitivity to ciprofloxacin, nitrofurantoin, oxolinic acid, and ofloxacin. However, resistance was observed against amoxicillin, lincomycin, spiramycin, tetracycline, colistin and cephalothin. Furthermore, PCR tests confirmed the presence of the aerolysin and hemolysin genes in the identified A. hydrophila strains. Sequencing the genes and conducting phylogenetic analysis based on the 16S rRNA sequence demonstrated that the A. hydrophila strain with H/A (accession no. OQ687115) had a 100% match