Two polyprenylated acylcyclopentanone racemates, evodialones A (1) and B (2), featuring a 3-ethyl-1,1-diisopentyl-4-methylcyclopentane skeleton, were isolated from an extract of the aerial parts of Evodia lepta. Evodialone A (1) was resolved by chiral-phase HPLC to afford a pair of enantiomers, (+)- and (-)-evodialones A (1b/1a), while evodialone B (2) could not be resolved. Their structures were elucidated by spectroscopic analysis and a combination of computational techniques including gauge-independent atomic orbital calculation of 1D NMR data and experimental and TDDFT-calculated ECD spectra. A putative biosynthetic pathway of 1 and 2 starting from the monocyclic polyprenylated acylphloroglucinols is proposed. All the isolates were screened for the antimicrobial activity in vitro, and 1a and 1b showed moderate inhibitory activities against several pathogenic fungi with MICs values of 17.1-68.3 μM.