A new α,β-unsaturated lactone, 2-(1-hydroxy-2-methylpropyl)-4-isopropylfuran-1(4H)-one (1), along with nine known compounds (2-10), was isolated from Aspergillus ochraceopetaliformis. The structure of compound 1 was elucidated based on comprehensive spectroscopic analysis, including 1D and 2D NMR (HSQC, COSY, HMBC), and HR-ESI-MS spectrometry. The absolute configuration was determined by comparison of the experimental and time-dependent density functional theory (TDDFT)-calculated electronic circular dichroism (ECD) spectra. Herein, we report the isolation, structure elucidation, and cytotoxic evaluation of the isolated compounds.