The aim of this study is to assess the toxicity of some heavy metals and parasitic infection in fishes as well as the immunological status of these fishes infected with different parasites. Between January 2018 and January 2019, 360 Nile tilapia (Oreochromis niloticus) near four farms and in the Nile River in Giza were examined for external and internal parasites. In addition, samples were expressed for two genes using quantitative real-time polymerase chain reaction. Samples were also taken from muscles and water for toxicological analysis of different heavy metals. In the skin, the mean TNF-α levels in the monogenea and mixed protozoan parasites (Trichodina and Myxobolus spp.) were 18.00 ± 0.67 and 13.65 ± 0.54, respectively. In the gills, the mean TNF-α of monogenea, Centrocestus formosanus, and encysted metacercaria (EMC) with monogenea group means was 20.45 ± 0.74, 23.00 ± 0.74, and 25.78 ± 0.74, respectively. In muscles with EMC, the mean TNF-α level was 14.67 ± 0.70. In livers with EMC, the mean TNF-α level was 26.78 ± 0.70. In the skin, the mean IL-1β level in monogenea and protozoan parasites (Trichodina and Myxobolus spp.) was significantly different (25.00 ± 0.69 and 15.00 ± 0.43, respectively). In the gills, the mean IL-1β level of the monogenean group was 21.00 ± 0.79, whereas C. formosanus showed the significantly highest value (27.00 ± 0.74). The mean IL-1β level of EMC with the monogenea group was 24.00 ± 1.54. Results showed that the level of Zn, Pb, Cu, and Cd was the highest in tissues of fish than permissible limits (PLs). Histopathological examination of different examined tissues exhibited serious pathological alterations. Monogenetic trematodes were detected in the examined fish, and their presence was accompanied by hyperplasia and fusion of the secondary gill lamellae. EMC of digenetic trematodes was frequently observed in the gills and in the cartilage rod. In conclusion, the parasite infection can upregulate the immunological cytokines with different levels with different infections.