The current study had aimed to assess the long-term dietary supplementation with Melaleucae aetheroleum, tea tree essential oil (TTO). The impact on growth performance, biochemical indices, immune function, oxidant/antioxidant activity, gene expression, histopathology, and resistance against Aeromonas sobria in Nile tilapia (Oreochromis niloticus) was investigated. Four groups (with five replicates; G1 (control group, G2, G3, and G4) of Nile tilapia received diets enriched with TTO (doses of 0.0, 0.5, 1.0, and 2.0 mL/kg diet) for 60 days, then fish were challenged by A. sobria. Outcomes indicated an extensive elevation in growth metrics (final body weight, weight gain, SGR, feed intake and fish body protein). Similarly, the total blood protein, albumin, total globulin levels, Serum complement-3 levels, lysozyme activity, immunoglobulin M (IgM), nitric oxide, and phagocytic activity were significantly enhanced in all treatments, notably in the 2.0 mL TTO/kg fed groups compared to the control. Lower levels of urea, creatinine, AST, ALP, ALT, cortisol, glucose, triglycerides, cholesterol and body crude lipids were observed in the fish that were fed a 2.0 mL TTO/kg diet. Supplementing TTO at 2.0-mL /kg diet revealed the best results for elevating CAT, SOD, and GSH activities plus declining MDA value in hepatic homogenate. Additionally, dietary 2.0-mL TTO/kg showed the best outcomes for the intestinal morphometry plus maintaining the histological picture in spleen and liver. Concurrently, fish that were fed a 2.0 mL TTO/kg diet exhibited a substantial upregulation of TNF-α, IL-1β, IL-10, TFG-β, IFN-γ and BCL-2 genes in the liver, while, caspase-3, and BAX were downregulated. Furthermore, TTO-enriched diets enhanced the relative percentage survival post-A. sobria challenge plus enhanced the clinical picture in a dose-dependent manner. Taken together, the findings revealed that long-term exposure to dietary TTO fortified the physiological performance, oxidant/anti-oxidant stability, immune function, gene expression, histological picture, and resistance of Nile tilapia against A. sobria.