This study explored the effects of mango seed (MS) powder supplementation on the growth, immune response, gene expression, and intestinal morphology of Nile tilapia (Oreochromis niloticus) over an 8-week period. A total of 300 Nile tilapia fingerlings (average weight of 15.29 ± 0.05 g) were divided into five treatment groups and fed either a basal diet or one of four experimental diets containing MS powder at concentrations of 10 (MS10), 20 (MS20), 40 (MS40), and 80 (MS80) g kg−1. The results demonstrated that Nile tilapia fed MS-supplemented diets experienced significant improvements (p < 0.05) in weight gain (WG), specific growth rate (SGR), and survival rate (SR) compared to the control group (0 g kg−1 MS). The MS-treated groups also showed a significant increase (p < 0.05) in the height and branching of intestinal villi along the entire length of the intestine, as well as a significantly higher villus-to-crypt depth ratio (V/C), indicating enhanced intestinal health and functionality. Moreover, although MS supplementation did not increase peroxidase activity, it did lead to a significant increase (p < 0.05) in the activity of skin mucus and serum lysozyme, along with upregulated gene expression of immune-related (IL-1, IL-8, and LBP) and antioxidant genes (GST-α, GPX, and GSR). Polynomial regression analysis identified an optimal MS dosage of 36.43–45 g kg−1 for effectively improving growth, immunity, and immuno-oxidant gene expression in Nile tilapia. These results emphasize mango seed (MS) as a promising natural supplement for improving the diet of Nile tilapia and, potentially, other freshwater fish widely used in aquaculture.