At the end of December 2022, a mortality outbreak was reported in net-cages farmed Nile tilapia in the state of São Paulo, Brazil, characterized by gross pathology consistent with bacterial hemorrhagic septicemia. This article presents the results of the study that aimed to; (a) isolate and identify the putative causative bacterial agent of the mortality outbreak by conventional isolation methods, DNA sequencing of specific 16S rDNA targets and housekeeping genes (gyrB and rpoB) and phylogenetic analysis; (b) evaluate the antimicrobial sensitivity and minimum inhibitory concentration of the causal agent to different antibacterial drugs to recommend optimal treatment of the disease under field conditions; (c) confirm Koch’s postulates and evaluate the pathogenicity of the causal agent by in vivo experimental infection challenge. In the current study, we have described the isolation, characterization, and virulence of the pathogenic agent, A. veronii, which caused hemorrhagic septicemia and mass mortalitu outbreak in tilapia farmed in Brazil. Overall, our results highlight the importance of effective epidemiological surveillance of diseases under field conditions to correctly identify the pathogens responsible for disease outbreaks and to promote appropriate and timely decision-making strategies to optimize the health and production management of the farmed tilapia industry and the prudent use of antimicrobials.