The goal of this study was to detect Aeromonas hydrophila and its antimicrobial resistance in Oreochromis niloticus sampled from the Nile River in Dakahlia Governorate, Egypt. A total of 150 moribund live and freshly dead tilapia fish (mean weight: 80 ± 10g, mean length: 20 ± 1.5cm) were collected during the 2022–2023 seasons. Fish samples were analyzed in the laboratory of the Fish Disease Research Department, Animal Health Research Institute, Dokki, Cairo. The clinical symptoms observed in the fish included surface hemorrhage, erosions, scale loss, and some samples showed exophthalmia, cataracts, and skin discoloration. Postmortem assessments revealed internal organ deterioration, yellowish fluid in the abdominal cavity, gallbladder enlargement, a pale liver, and pigmentation on the liver's surface. A. hydrophila identification was based on phenotypic features and homology of 16S rRNA gene sequences. The 16S rRNA gene of the isolates was submitted to GenBank under accession number PP829284.1, showing high nucleotide identity with other Egyptian isolates. Antimicrobial sensitivity testing indicated that the A. hydrophila isolates were sensitive to ciprofloxacin, nalidixic acid, oxolinic acid, amikacin, ofloxacin, flumequine, and nitrofurantoin, but resistant to tetracycline, lincomycin, gentamicin, amoxicillin/clavulanic acid, erythromycin, trimethoprim/sulfamethoxazole, and oxytetracycline. Genetic characterization confirmed the presence of aerolysin genes and resistance genes blaTEM and tetA, while hemolysin, ermB, and mcr1 genes were absent. These findings highlight the need for effective monitoring and management strategies to control the A. hydrophila pathogen in aquaculture.