Peribysins O (1), P (3), and Q (4) were isolated from Periconia macrospinosa KT3863. The relative configuration of the 6,7-epoxide of 1 was elucidated by performing quantitative NOE experiments. The structure of 2, which is a tautomer of 1 present in CDCl3 solutions in 5% abundance, was also fully characterized by NMR analysis. Their absolute configurations were independently determined by the modified Mosher's method (for 1 and 3), the electronic circular dichroism (ECD) exciton coupling theory after conversion into dibenzoate 9 (for 3), and theoretical ECD calculations (for 1, 3, and 4). The obtained relative structures 1, 3, and 4 were verified by calculating their 13C chemical shifts using density functional theory (DFT). Although the established (4 S)-enantiomer for 1-4 is in accordance with that of other peribysins isolated from the related fungus Periconia byssoides OUPS-N133, Danishefsky's total synthesis of peribysin E (5) led to the subsequent revision of the (2 R,4 S,5 R,6 S,7 S,8 R,10 S)-enantiomer to the (2 S,4 R,5 S,6 R,7 R,8 S,10 R)-enantiomer. This discordance led us to reinvestigate the configuration using time-dependent DFT-based ECD spectral calculations, which supported the original (4 S)-enantiomer.