The development of nanoemulsions has garnered interest for their potential applications. Currently, there is no data on the effects of chitosan grape seed oil nanoemulsion (CGON) on Nile tilapia. Consequently, this study intended to investigate the impact of CGON supplementation on growth performance, digestive enzymes, immune-antioxidant, and hepato-renal biomarkers of Nile tilapia. Following a 60-day feeding period, a bacterial challenge with Aeromonas veronii (A. veronii) was implemented, and tissue samples were collected to evaluate changes in histopathology and immune transcriptomics. There were 240 Nile tilapia fish (24.02 ± 0.18 g) spread out across twelve glass aquariums with a concentration of 15 fish per tank. A fully randomized design was employed in four replicates, with the control group receiving 0 mg /kg CGON (CGON0), while CGON10, CGON20, and CGON40 were administered 10, 20, and 40 mg /kg diet CGON, respectively. Dietary CGON supplementation significantly enhanced growth performance, as indicated by improved feed intake, specific growth rate, weight gain percentage, final weight, and increased serum amylase and lipase activities, with the highest performance observed in fish receiving 40 mg/kg CGON. Increasing levels of CGON in fish diets were found to cause notable improvements in hepatic antioxidant, and blood immunological indices. In fish fed 40 mg/kg CGON, total protein, globulin, and albumin levels increased, while markers of hepato-renal stress were reduced. After A. veronii challenge, the expression of immune-related genes (TP4, TP3, β-defensin-1, CSF1R, hepcidin-2, IL-10, and TGF-β) was significantly upregulated in fish supplemented with CGON. Histopathological examination revealed noticeable tissue damage in the CGON0 group, whereas fish receiving 40 mg/kg CGON showed less severe abnormalities. In conclusion, the inclusion of CGON in fish diets was shown to enhance growth performance. A dietary level of 40 mg/kg CGON was recommended for O. niloticus, as it improved antioxidant status, immune response, and disease resistance.