AbstractArundifungin (1) has been reported as a potent antifungal agent against Candida and Aspergillus spp; however, only its planar structure has been disclosed. This paper describes the assignment of the relative and absolute configuration of 1, which includes (a) determination of the relative configuration of the ABCD polycyclic ring moiety on the basis of detailed nuclear magnetic resonance (NMR) analysis, followed by the confirmation with density functional theory (DFT)–based 13C NMR chemical shift calculations, (b) determination of the absolute configuration of the ABCD polycyclic ring moiety by observing a positive Cotton effect at 217 nm because of the C‐8/C‐9 tetrasubstituted double bond and its reproduction using DFT calculations, (c) determination of the configurational relationship between C‐17 and C‐20 by a combination of nuclear Overhauser effect (NOE) analysis and DFT‐based conformational analysis, and (d) determination of the relative and absolute configuration of the C‐24 and C‐25 asymmetric centers on the acyclic side chain by a combination of chemical derivatization including modified Mosher's method and DFT‐based conformational analysis, followed by electronic circular dichroism (ECD) spectral reproduction. Present study also discovered 26‐deoxyarundifungin (2) of which relative structure was readily elucidated by 1H and 13C spectral comparison with those of 1. Since 2 exhibits slightly weaker antifungal activity against Cochliobolus miyabeanus than 1, the hydroxy group at C‐26 moderately contributes to the activity.