This study aimed to investigate the relationship between the ectoparasite infestation of O. niloticus with gene expression analysis and its histopathological alteration. Thus,  from two localities in Egypt, a total of 423 O. niloticus fish were collected and subjected to an investigation during the period from October 2022 to July 2023. Fish were surveyed for ectoparasites. Samples from gills were collected and fixed in 10% neutral buffered formalin. The total RNA was extracted from both the skin, gills, Cpy1a1 and TNF-α were analyzed. The examined fish showed skin hemorrhage, ulcers, sloughed scales, and corneal opacity. Fish were found to have Clinostomum spp < em>. on the skin and gills and Lamproglena parasitic crustacea were attached to gills.  From 423 examined O. niloticus fish, 73 fish specimens from various locations in Egypt were examined for their rate of ectoparasites occurrence. Infestation in fishes of the River Nile branch (68.5%) was higher than in cultured fish (57.1%). The rate of infestation was 200 fish with monogenean; 50 fish with protozoa, and 10 fish by crustacean, while the mixed infection was detected in 10 fish individuals with crustacean and monogenean; 3 fish with crustacean and protozoa; 50 fish with monogenean and protozoa, and 5 fish with three types of these ectoparasites. Two protozoan parasites, Trichodina heterodentata (T.heterodentata), and Myxobolus tilapiae (M. tilapiae)  were among the various ectoparasites found in the examined fish as well as Centrocestus formosanus (C. formosanus); Cichlidogyrus tilapiae (C. tilapiae)and Lamproglena monodi (L. monodi). Histopathological examination of gills revealed the presence of parasitic infestation between gill filaments; some cases showed heavy infestation with multiple parasitic worms. Gene expression analysis showed up-regulation of the Cpy1a1, and TNF-α in the M and L groups compared to the negative control group in both skin and gills. Treatment of both infested groups recorded down-regulation of the tested genes in the skin. However, the L-treated group showed a non-significant decrease in the expression level relative to the L group in the gills.