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Endophytic fungi were first isolated from the fresh root of the Chinese medicinal plant <i>Vernonia anthelmintica</i> collected from the Hotan Prefecture within the Xinjiang Autonomous region of the People’s Republic of China. This plant has been used in Uyghur traditional medicine to treat vitiligo, a skin condition characterized by patches of the skin losing their pigment. In total, fifteen fungal strains were isolated. Among these, four endophytic fungi were identified by their DNA sequences and registered to GenBank with accession numbers. The isolates were identified as <i>Schizophyllum commune</i> XJA1, <i>Talaromyces</i> sp. XJA4, <i>Aspergillus</i> sp. XJA6, <i>Aspergillus terreus</i> XJA8. Ethyl acetate extracts of all fungal strains were used to quantify melanin content and to identify in vitro biological activity assays including antimicrobial, antioxidant, cytotoxic, antidiabetic and tyrosinase activity on B16 cells. Among the extracts of all four identified strains, the ethyl acetate extract of the <i>Aspergillus</i> sp. XJA6 was chosen for further characterization because it presented the highest biological activity against these tests. In addition, twenty four volatile compounds from the petroleum ether fraction were characterized by GC–MS.