Vibriosis is a common disease in aquaculture. Nile tilapia (Oreochromis niloticus) farmed within some commercial fish farms in Kafr El-Sheikh governorate Egypt showed signs of disease and exhibited heavy mortality. In order to get to the root of the problem, ninety moribund tilapia were sampled from the affected fish farms and subjected to bacteriological and molecular examinations. Biochemical characterization of bacterial isolates was performed via the API 20E analytical system. All examined fish samples were infected with Vibrio species. Eighteen Vibrio isolates, V. mimicus (n = 12), and V. cholerae (n = 6) were taken randomly for molecular characterization and further analysis. Isolates were genotyped via sequencing and alignment of the recA gene. Isolates possessed numerous virulence traits, including the production of hemolysins, proteases, lipases, and nucleases. The prevalence of ompU, vmh, vpsR, and flrA virulence genes in Vibrio strains was 61.1%, 66.6%, 27.7%, and 33.3 %, respectively. The blaTEM (55.5%), apHAI (50%), and qnrVC (27.7%) antibiotic resistance genes were recorded in Vibrio strains. All Vibrio isolates (100%) were resistant to ampicillin 10 μg and amoxicillin 30 μg, while they showed high sensitivity against florfenicol 30 µg (83.3%) and ciprofloxacin 5 µg (77.7%). Challenge experiments in Nile tilapia confirmed pathogenicity of the isolates. Fish showed symptoms of septicemia and high mortality was observed. Infections induced numerous histopathological alterations in diseased fish. This is the first report of V. mimicus outbreaks associated with mass mortality in Egyptian farmed Nile tilapia. The findings form the basis for future development of effective control and preventive measures against one of the most infectious pathogens that affect fish and humans.