The herbicidal mechanism of the components extracted from Pythium aphanidermatum was examined in this study. Component I was isolated using the HPD500 macroporous adsorption resin and HPLC. Its impact on seed germination and plant growth of weeds was determined and the contents of MDA, superoxide anion radical, and the activities of hills and roots were examined. The root length of weed plants was inhibited under illumination while the stem height was inhibited evidently under darkness. The relative electric conductivity of Digitaria sanguinalis and Amaranthus retroflexus under illumination was 94.55 μS-cm-1 and 58.75 μS-cm-1, respectively, whereas that under darkness was 85.25 μS·cm-1 and 36.25 μS·cm-1, respectively. The MDA contents of Digitaria sanguinalis and Chlorella pyrenoidosa were 0.08385 μmol · L-1 and 0.1742 μmol · L-1 under illumination, respectively, while those were 0.0129 μmol·L-1 and 0.01935 μmol ·L-1 under darkness, respectively. Simultaneously, superoxide anion radical content was higher under illumination than under darkness. These results showed the photosynthesis was affected by component I extracted from Pythium aphanidermatum.