The River Nile in Egypt is the most important natural freshwater fishery that can secure thousands of tons of fish. Aeromonas hydrophila is a common pathogen that can induce significant deaths in African catfish (Clarias gariepinus). During the summer of 2022, 100 moribund and freshly dead African catfish were collected from the Nile River, Al-Manial, Cairo, Egypt for a bacteriological examination. The clinical findings of surface hemorrhage and abdominal distention hemorrhage at the anal opening resulted in skin discoloration and loss of antenna. The postmortem examinations included the deterioration of internal organs, hepatomegaly, and splenomegaly. The isolated A. hydrophila strains were identified based on phenotypic characterization by diagnostics SRO. GN24 features and the homology of 16S rRNA gene sequence analysis. Antibiogram has revealed that isolates were sensitive to gentamicin, ciprofloxacin, nitrofurantoin, nalidixic acid, oxolinic acid, and ofloxacin, while they were highly resistant to amoxicillin, lincomycin, spiramycin, colistin, tetracycline, and cephalothin. In addition, PCR data confirmed the presence of the Aerolysin gene in the identified A. hydrophila isolates, while negative to the Hemolysin gene. Gene sequencing and phylogenetic analysis based on the 16S rRNA sequence confirmed that A. hydrophila H/A (accession No. OL771444) of the present study displayed 100% identity with the 16S rRNA gene of A. hydrophila. The presence of resistance genes was investigated on either the bacterial DNA or the plasmid using a plasmid curing method, which was achieved by elevated temperatures. In conclusion, A. hydrophila is the main cause of mortality in C. gariepinus, and the wise use of antibiotics in fish treatment has become compulsory to avoid antibiotic residues and the great losses due to multidrug resistance (MDR).