One of the most significant rivers in the world is the River Nile. It is the source of 96% of the country's renewable freshwater. The study of freshwater fungi is important because it reveals the hidden diversity and ecological significance of these organisms in aquatic ecosystems. It deepens our understanding of biodiversity, nutrient cycling, ecological interactions,  and water quality, while also offering practical applications for environmental and biotechnological purposes. During surveys for freshwater ascomycetes in the Nile River as well as Ibrahimia and Fayama Canals at Assiut region, Egypt, ten species belonging to six genera of families: Chaetomiaceae, Podosporaceae, and Lasiosphaeriaceae, were isolated and identified. Among them, Botryotrichum geniculatum, B. trichorobustum, Pseudorhypophila marina, Pseudothielavia terricola, Zopfiella indica, and Z. udagawae were reported in the current study for the first time from Egypt. These species were isolated in pure cultures and identified based mainly on their morphological features. The identification of one species was confirmed genetically and identified as Pseudothielavia terricola AUMC 16038 (OQ581575). The treated species were described, and photographic illustrations were captured. The frequency, distribution, and hosts for each listed species were recorded. Seven out of ten fungal species have been recovered from Phragmites australis, which is considered in this study to be the ideal submerged plant for isolating freshwater ascomycetous fungi. Continued research on freshwater fungi is of paramount importance, and it is crucial to prioritize conservation efforts to protect rare and endangered species.