Waterborne trace element mixture (TEM) negatively affects the physiological performance of fish and health status in recent years. Pumpkin peels (Cucurbita pepo, CPP; 12 g/kg diet) as a target of polyphenol substances was evaluated against TEM [mercury (Hg) and zinc (Zn)] exposure in Oreochromis niloticus (Nile tilapia) in this investigation. Comprehensive biochemical, molecular, and histological assays were evaluated. A total of 160 fish (28.50 ± 1.33 g) were assigned into four groups for 60 days. The control and CPP groups were fed basal and CPP diets without trace element exposure, respectively. The TEM and CPP + TEM groups were exposed to TEM (Hg at 0.05 mg/L and Zn at 7.00 mg/L) and given the basal and CPP diets, respectively. Fish mortality (27.50%) and hepato-renal variables increased significantly in TEM fish. The TEM group experienced lower serum complement 3 activity, lysozyme activity, and nitric oxide levels. Furthermore, downregulation of immune genes (interleukin-10, heme oxygenase-1, and Kelch-like ECH-associated protein-1) expression and upregulation of tumor necrosis factor-α gene expression were observed in the TEM group. Several pathological lesions were observed in the liver, kidney, and splenic tissues of the TEM fish. Fortification with CPP in the diet decreased the mortality rate and modulated hepato-renal markers in the TEM group. The humoral and splenic immune responses of the TEM fish and histological architecture were enhanced with the CPP-supplemented diet. Overall, the dietary intervention of CPP (12 g/kg diet) is recommended to overcome TEM negative effects on the physio-biochemical and immunological responses of Nile tilapia.