Fungi belonging to the genus Aspergillus were first described 300 years ago by the botanist Antonio Micheli. Aspergillus spp. genomics has been intensively investigated, and over 40 genomics analyses have been reported for these fungi. Aspergillus species are cosmopolitan and are biochemically and physiologically versatile, growing in an array of growth media and expressing the secondary metabolism for tenths of metabolites [1,2]. Few phenyl-a-pyrones, including campirones A-C [3], pyrophen [4,5], isopyrophen [4] and naphthoquinoneimine [5,6], have been reported as fungal metabolites. An Aspergillus sp. DLM38 strain obtained from the ascidian Didemnum ligulum produces in growth media two phenyl-a-pyrones, (-)-naphthoquinoneimine (1) and pyrophen (2). Feeding experiments with [1-13C]acetate, [1,2-13C2]acetate, [3-13C]-ʟ-phenylalanine and [1-13C]-ᴅ-glucose were performed in order to investigate the biosynthesis of (-)-naphthoquinoneimine and pyrophen. Analysis by UPLC-QToF-MS/MS and 13C-NMR of [1-13C]-ᴅ-glucose labeled compounds enabled us to unambiguously establish the biosynthesis of both compounds, which is organized by a NRPS-PKS machinery.