Nile tilapia aquaculture is a fast-growing industrial sector in Egypt. However, the progress of this industry is hindered by many challenges as poor water quality and associated bacterial infections. Aeromonas hydrophila is an important zoonotic waterborne aquatic pathogen responsible for severe outbreaks in tilapia culture so the current study aimed to investigate the prevalence of Aeromonas hydrophila in tilapia fish and their aquaculture water, the fish and water samples were collected from three farms located in Assiut and Minia Governorates, Egypt. Aeromonas hydrophila was isolated from tilapia five organs including liver, kidney, intestine, spleen and gills with a percentage of 7.1%, 6.5%, 9.1%, 5.1%, 11.7%, receptively, and from water samples with 59.7%. The isolates were molecularly confirmed as Aeromonas hydrophila in 70.9% and 82.4% of the tested isolates using 16s RNA and gyr-β primers, respectively. Aeromonas hydrophila isolates revealed a marked resistance for the tested antibiotics; amoxicillin and novobiocin (100%), streptomycin (71.4 %), chloramphenicol (57%), doxycycline and trimethoprim/Sulphamethoxazole (50%), colistin (43%), ciprofloxacin and norfloxacin (14%). The isolates showed multiple antibiotic resistance indexes ranging from 0.3 to 1. The present study highlights the Aeromonas hydrophila resistance and virulence, tilapia aquaculture health hazard to the human population, so adequate control measures should be applied. Furthermore, there is an essential need to promote an alternative non-antibiotic control in farmed fish.