Pythium aphanidermatum was cultured in PD medium and the mycelia were extracted with ethyl acetate and methanol, and the methanol extracts were separated gradually by silica gel column using ethyl acetate mixture and petroleum ether (V/V=3:1 and V/V=2:1) as the developers. Eluents were collected and each 50 mL were considered as a fraction and bioassayed. The results revealed that fraction 21?24 showed strong activity against Digtaria sanguinealis with the inhibition level of 4. The combined fractions of 21?24 were eluted with the mixture of ethyl acetate and petroleum ether (V/V=2:1) as the developer and 20 fractions were collected. Bioassay results showed that fraction 3 had the stronger inhibitory activity against Digtaria sanguinealis. HPLC analysis suggested that this fraction mainly contained 3 components, the retention time of which was 12.7, 14.0 and 30.5 min respectively.