In summer 2021, acute mortalities occurred in Nile tilapia farm in Qena governorate. Different hypothesis including the implication of environmental factors, and/or pathogens have been explored. Clinical, parasitological and bacteriological examinations have been performed. In addition, the water quality parameters were assessed. The clinical investigation of the moribund and recently dead fish demonstrated generalized septicaemia with high mortality rates (67%). The phenotypic and biochemical characterization of the pure bacterial colonies that isolated from 60 moribund fish revealed Gram-negative rods and identified as Aeromonas species. The identity of the isolates was identified as Aeromonas species by amplification of Aeromonas 16S rDNA gene fragment by PCR and confirmed as Aeromonas veronii by amplification and sequencing the Aeromonas gyrB-gene. The water quality parameters showed an increase in the pH and ammonia levels over the permissible levels. Experimental infections were performed to confirm the virulence of the isolated bacteria. Results revealed that A. veronii isolate produced the same clinical picture of the collected samples with mortality rate 78 %. The present study implicates that A. veronii could be the causative agent of the Nile tilapia mortality in Qena, without neglecting the role of water quality in worsening this outbreak.