AbstractGrowing Nile tilapia in brackish water showed promising results, but the possibility of ammonia exposure can interrupt health status and productivity. Herein, the study tested the combined effects of water salinity and ammonia exposure on the antioxidative status, serum biochemistry, and immunity of Nile tilapia. Fish were assigned to eight groups where fish were reared in saline water (5, 10, and 15 ppt) with continuous or intermittent (every 3 days) total ammonia (TAN) exposure (5 mg TAN/L) (2 × 4 factorial design). After 30 days, the water salinity, TAN, and their interaction were markedly (P < 0.05), affecting the growth performance (final weight, weight gain, and specific growth rate) and survival rate of Nile tilapia. The growth performance and survival rate were markedly lower in tilapia grown in 15 ppt with continuous TAN exposure than in the remaining groups. The results showed that fish exposed to higher salinity levels (10 and 15 ppt) and continuous TAN exposure had a more robust antioxidative response, as evidenced by higher superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities and lower malondialdehyde (MDA) levels in the homogenates of the gills, intestines, and livers. The gills were notably affected, with congestion of primary filaments blood vessels and degeneration or shedding of secondary filaments epithelium, especially at salinity levels of 10 and 15 ppt. Additionally, the intestines displayed hyperplasia and inflammatory cell infiltration of intestinal mucosa at 5–10 ppt salinity, degeneration and sloughing of the intestinal epithelium at 15 ppt saline water, and increased goblet cell number at salinity of 10 ppt. The study found that continuous TAN exposure had a more significant impact on the fish, especially at higher salinity levels. Water salinity, TAN, and their interaction significantly affected all measured blood bio-indicators (total, albumin, globulin, ALT, AST, creatinine, urea, glucose, and cortisol levels). The phagocytic activity and index were markedly lowered in fish reared in 15 ppt with continuous TAN exposure, while the lysozyme activity was decreased in fish grown in 5, 10, and 15 ppt with continuous TAN exposure. In conclusion, Nile tilapia showed the possibility of growth with normal health status in brackish water (5–10 ppt); however, continuous TAN exposure can impair the productivity of tilapia, especially with high salinity (15 ppt).