The accumulation potency of zinc nanoparticles in Nile tilapia (Oreochromis niloticus) was previously studied but their impacts on proximate chemical composition in muscle tissue by describing the dose-dependent accumulation and the protective role of vitamins (E + C), have not been investigated. Therefore, this study was carried out to assess the protective role of vitamins (E + C) on Zn accumulation in muscle and gill tissues of O. niloticus exposed to three sub-lethal concentrations (1/8 LC50, 1/4 LC50, and 1/2 LC50) of zinc oxide nanoparticles (ZnO NPs) compared to zinc oxide bulk particles (ZnO BPs) as well as their effects on the induced chemical composition alterations for different experimental periods (7, 14, 21, and 28 days). The data displayed that fish exposed to the different sub-lethal concentrations of ZnO NPs or ZnO BPs have a significant increase (P<0.05) in Zn ions accumulation in muscle and gill tissues compared to control group but Zn was accumulated in gill tissue higher than muscle tissue at all exposure periods. Also, Zn accumulation was higher in fish tissues exposed to ZnO NPs than ZnO BPs. On the other hand, groups supplemented with vitamins (E + C) showed a significant decreasing (P<0.05) in accumulated Zn levels compared to groups without supplementation. The values of these supplemented groups returned to similar levels established in the control at low concentrations but still higher than control at the high concentrations. Furthermore, the results showed that moisture and ash content slightly increased while protein and fat decreased in fish exposed to ZnO NPs or ZnO BPs compared to control group. In conclusion, the findings supported that a combination of vitamins (E + C) reduced Zn accumulation and ameliorated chemical composition alterations in O. niloticus fish.