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Hyoscyamus muticus L. (Egyptian henbane) is one of the desert medicinal plants of family Solanaceae. The plant produces pharmaceutically important compounds (tropane alkaloids) as secondary metabolites. In the present study, we describe mycoflora of H. muticus grown in four different locations in Egyptian southern desert (Aswan region): Aswan university campus, Wadi Allaqi down stream part, Aswan airport road, and Sahari city. Eighty-one species and two varieties belonging to 31 genera were isolated from soils surrounding H. muticus plants, the surface of the plants, and inside the plants as endophytic fungi. Aspergillus was the most common genus in all study areas. The highest number of genera and species of fungi were recorded in Aswan university campus followed by Aswan airport road. Fungal diversity analysis revealed that these two locations have higher fungal diversity compared to other two locations. A higher number of fungal species were isolated from rhizosphere soil and rhizoplane than from non-rhizosphere and other plant organs. Endophytic fungi were isolated from all plant parts of H. muticus. Communications between H. muticus plants and fungi under desert conditions both in rhizosphere and inside the plant are deduced.