Exophialin, a previously unknown indole alkaloid, was isolated from the human-pathogenic fungus Exophiala dermatitidis upon cultivation on a medium containing tryptophan as sole nitrogen source. Its structure has been elucidated by 1D and 2D NMR experiments and mass spectrometry. When the mutant strain Mel-1 was used in place of the wild-type strain, the new indole alkaloid 8-hydroxyexophialin was generated instead of exophialin. This result indicates that the aromatic moiety of exophialin originates from 2-hydroxyjuglone, a shunt product of the 1,8-dihydroxynaphthalene melanin biosynthesis, which is only produced by the wild-type strain, but not by the mutant. In contrast, the mutant accumulates flavioline, which acts as precursor leading to the generation of 8-hydroxyexophialin instead of exophialin.