In this study, chemical compositions of fatty oils and bioactivity of crude extracts from Holotrichia diomphalia larvae as Chinese materia medica were investigated for the first time. The chemical compositions of the fatty oils were obtained by two different methods and determined by GC/MS. In total, the petroleum ether extract produced 21 compounds (96.3%) while the supercritical fluid extract produced six compounds (99.53%) for identification. The effect of petroleum ether and other crude extracts on Pyricularia oryzae was also examined. Results indicated that ethanol and petroleum ether extracts had excellent antifungal activities. These findings demonstrated that fatty oils from H. diomphalia larvae had great potential to be used as a source for natural health products.