Aster tataricus is a plant used in Traditional Chinese Medicine. From its roots, we isolated four endophytic fungi strains. After mass spectrometry analysis and subsequent molecular networking and dereplication, one of the strain’s extracts showed a cluster of yet undescribed natural products. Additionally, the extract was found to be lethal for the nematode Caenorhabditis elegans and cytotoxic against eukaryotic cell lines. The fungal strain was characterized by morphological and molecular studies, allowing its description as a new species in the genus Tengochaeta (Chaetomiaceae), Tengochaeta bulbillosa. After cultivation and extraction of the strain, the major secondary metabolites were isolated. Structure elucidation based on nuclear magnetic resonance spectroscopy and high-resolution tandem mass spectrometry revealed these compounds to be five new azaphilones. Additionally, the localization of these azaphilones in the host plant was studied by mass spectrometry imaging of different plant tissues, revealing that they were mainly localized in the aerial parts of the plant. The main compound, bulbillosin A, was evaluated for its activity against sixty cancer cell lines, revealing a differential cytotoxicity profile.
