A new antifungal butenolide irperide (1) along with five known compounds were isolated from the co-culture of endophyte Irpex lacteus and pathogenic Nigrospora oryzae. The structure of 1, including the absolute configuration, was elucidated by analysis of NMR, HR-ESI-MS data and ECD spectra. Compounds 1, 4 and 6 exhibited significant antifungal activity against Aspergillus fumigatus, with MIC values of 1, 2 and 1 μg/mL, respectively.