The effects of long-term dietary supplementation with sandalwood (Santalum album L.) essential oil (SEO) was investigated on hemato-biochemical biomarkers, immune status, antioxidant capacity, and resistance against Staphylococcus aureus in Nile tilapia (Oreochromis niloticus). Five groups (with four replicates) of O. niloticus (12.60 ± 0.20 g) were fed diets supplemented with SEO at doses of 0, 0.5, 1.0, 2.0, and 4.0 mL/kg diet for 60 days. Results indicated a substantial increase in blood protein levels and lower serum cholesterol, cortisol, glucose, urea, creatinine levels and, transaminase activities of fish fed a 2.0-mL SEO/kg diet. Serum lysozyme activity, nitric oxide, complement-3 levels, and phagocytic activity were significantly improved in O. niloticus after 60 days of feeding SEO-supplemented diets. Dietary SEO at level of 2.0-mL SEO/kg diet increased the activities of SOD, CAT, and GPx, and decreased MDA levels in liver homogenate. In addition, dietary 2.0-mL SEO/kg diet significantly upregulated antioxidant genes expression (CAT, SOD, GPx, GST, and GSR) with downregulation of apoptotic genes (HSP70, TLR2, caspase-3, and PCNA) in the liver. Furthermore, SEO-enriched diets significantly down-regulated pro-inflammatory (TNF-α, IL-1β, and IL-8) and up-regulated anti-inflammatory cytokine genes (TFG-β and IL-10) in the spleen. Moreover, SEO fortification increased the relative percentage of survival against S. aureus challenge and regulated immune-antioxidant genes in the spleen after the challenge. Overall, the results revealed that long-term using SEO might strengthen the physiological performance, hepatic oxidant/antioxidant balance, innate immune response, and resistance of O. niloticus against bacterial infections.