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Fish are a source of high-quality protein with low cholesterol, but they are susceptible to parasitic infections, which have a significant impact on aquaculture, in addition to their zoonotic potential. The present study estimated parasitic infections and evaluated the diversity of zoonotic parasites in freshwater Nile tilapia (<i>Oreochromis niloticus</i>) in Assiut Governorate, Upper Egypt. A total of 300 samples were randomly collected from the Assiut Governorate. These fish were examined for both ectoparasites and endoparasites, followed by the experimental infection of mice with encysted metacercariae (EMC) for the retrieval of the adult worms. The overall prevalence of the variable parasites was 82% (246 of 300). Both ecto- and endoparasites were detected in 41% (123 of 300) of the examined fish. The identified ectoparasites were <i>Gyrodactylus</i>, <i>Dactylogrus</i>, <i>Cichlidogyrus</i>, <i>Trichodina</i> and <i>Icthyophthirius multifiliis</i>, in 5%, 4%, 22%, 6% and 4% of the fish, respectively. The endoparasites were trematodes <i>(Orientocreadium batrachoides</i> 3%), nematodes (<i>Contracaecum</i>. 2%), acanthocephala (<i>Acanthosentis tilapiae</i> 25%) and protozoa that included <i>Isospora</i> and <i>Eimeria</i> spp., in 1% and 8% of fish, respectively. <i>Myxobolus</i> was detected in 2% of the examined fish. The overall prevalence of encysted metacercariae (EMC) was 95% (285 of 300), while infection with macroscopic EMC had a prevalence of 37% and microscopic EMC had a prevalence of 58%. The adult worms recovered from the experimental infections were <i>Prohemistomum vivax</i> and <i>Mesostephanus</i> spp., which belong to the family <i>Cyathocotylidae</i>. Collectively, these findings reflect the relatively high occurrence of parasites among the studied fish, confirming the necessity of strict measures to control infection.