Functional trace elements and vitamins can boost immunity and antioxidative response in aquatic animals without creating environmental hazards. While nano-selenium (Nano-Se) and vitamin C (VC) have been used as immunomodulators and antioxidants in animal and poultry feed, there is little data on Nano-Se and/or VC supplementation in aquatic animals. Thus, the current study evaluated the impact of adding Nano-Se and VC to the diets of Nile tilapia for 8 weeks. Four diets were formulated and offered to the fish: no supplementation (control), 1 mg Nano-Se/kg, 500 mg VC/kg, and 1 mg Nano-Se + 500 mg VC/kg of food. Growth-related parameters (final body weight, weight gain, and specific growth rate) were significantly increased in tilapia fed Nano-Se and VC, with a reduced feed conversion ratio (P < 0.05). Intestinal villus length and width as well as the number of goblet cells were increased in tilapia fed Nano-Se and/or VC (P < 0.05). Additionally, dietary Nano-Se and/or VC significantly increased nitro-blue tetrazolium (NBT) level, superoxide dismutase, glutathione peroxidase, catalase, the phagocytic index, and lysozyme and phagocytic activities (P < 0.05). However, significantly reduced levels of malonaldehyde were observed in fish fed Nano-Se and/or VC (P < 0.05). TNF-α and IL-1β gene expressions in the liver and spleen of the fish were significantly upregulated by Nano-Se and/or VC (P < 0.05). The results revealed the potential role of Nano-Se and/or VC in enhancing growth, intestinal morphometry, and immune and antioxidative responses in Nile tilapia.