The structure of a new cytochalasin, ascochalasin, isolated together with deoxaphomin from Ascochyta heteromorpha (pathogen of oleander), was determined mainly by ¹H-nmr and ¹³C-nmr spectroscopy. A previous analysis of the organic culture filtrate extracts of A. heteromorpha led to the isolation of cytochalasins A and B; further investigation revealed two additional cytochalasins: deoxaphomin (known) and the new ascochalasin. The isolation involved chromatographic purification using SiO₂ columns and preparative thin-layer chromatography (SiO₂ and reversed-phase plates). Ascochalasin, an amorphous solid with molecular formula C₂₉H₃₉NO₄ (from high-resolution mass spectrometry), was structurally characterized by comparing its ¹H-nmr (including decoupling experiments) and ¹³C-nmr data with those of deoxaphomin and cytochalasin B, analyzing its diacetyl derivative, and identifying key differences (e.g., ascochalasin lacks a double bond at C-21/C-22, resulting in aliphatic C-21/C-22 methylene groups). The diacetyl derivative confirmed hydroxyl groups at C-7 and C-20. In conclusion, the structure of ascochalasin is (7S, 13E, 16R, 20R)-7,20-dihydroxy-16-methyl-10-phenyl-[13]cytochalasan-6(12),13-diene-1,23-dione. The biological properties of cytochalasins suggest ascochalasin will be tested in phytotoxic, mycotoxic, and antibacterial bioassays.