AbstractFungi of the genus Sepedonium (anamorphic ascomycetes) are known to infect fruiting bodies of Basidiomycetes of the order Boletales. We have characterized twelve Sepedonium isolates by intact‐cell mass spectrometry (IC‐MS) with the help of respective biomarkers and their metabolite spectra focusing on peptaibol production. A strain of mycoparasitic S. chalcipori was grown in solid‐state fermentation, and tylopeptin production was found, suggesting an ascomycete origin of these peptaibols, which were first described in the basidiomycete Tylopilus neofelleus. In addition, the structures of two new peptaibols, chalciporin A (=Ac‐Trp‐Val‐Aib‐Val‐Ala‐Gln‐Ala‐Aib‐Ser‐Leu‐Ala‐Leu‐Aib‐Gln‐Leuol) and chalciporin B (=Ac‐Trp‐Val‐Aib‐Val‐Ala‐Gln‐Ala‐Aib‐Gln‐Aib‐Ala‐Leu‐Aib‐Gln‐Leuol) are presented. The IC‐MS technique was applied for in situ peptaibol analysis of Sepedonium strains growing on Boletales, in particular S. chrysospermum infecting Xerocomus cf. badius. We found chrysospermins at the surface and within basidiomycete tissue, as well as in the cultivated parasite.