HYOSCYAMUS muticus L. (Egyptian Henbane), a shrub thriving in the sandy regions of Egypt, shows important pharmacological properties which may be attributed to its own bioactive compounds or due to associated endophytes. In the present study, roots, stems and leaves of H. muticus growing in Egypt were evaluated for the presence of associated fungal endophytes, and secondary metabolites produced from them especially the characteristic plant tropane alkaloid; hyoscyamine. Using culture-dependent approach, sixteen fungal endophytes belonging to five different genera were identified morphologically according to their culture appearance and detailed microscopic structures. The isolated endophytic fungi were cultured using three different media (PDB, RG and SDB) and each filtered broth was sequentially extracted with ethyl acetate. Crude extracts collected from different fungal isolates were subjected to phytochemical analysis using thin layer chromatography (TLC) which showed a distinct fingerprinting profile of several fungal secondary metabolites. The total secondary metabolite, phenolic compound, flavonoid, anthraquinone glycoside, coumarins and alkaloid content was investigated. Different activities of the used media were reported on the changing secondary metabolic profile of each fungus. Furthermore, the ability of the tested fungal endophytes to produce hyoscyamine, characteristic to H. muticus was tested by TLC analysis. Some isolated endophyte species belonging to Aspergillus and Penicillium showed positive tests for hyoscyamine.