In order to clarify the mechanism of herbicidal activity of strain HY-021 on Chenopodium album L., electron microscope scanning technology was used to observe the process of infestation of C. album leaves by the strain after inoculation, and changes in the activity content of C. album plants after inoculation were determined using physiological index assay. The results showed that the mycelium of the HY-021 strain invaded leaf tissues through stomata, parasitized, proliferated and produced spores, which gradually destroyed the tissue. Concurrently, leaf surfaces began to show chlorosis and developed disease spots, ultimately leading to plant death due to an inability to perform photosynthesis. Following inoculation of C. album plants with the HY-021 strain, the content of MDA inside the plant bodies gradually increased, while the activities of enzymes such as SOD, CAT, and POD showed trend of increasing and then decreasing after inoculation. It showed that the strain had multiple effects on the plant physiological mechanisms and defense enzymes, and caused a wide range of metabolic changes in the plant after inoculation. The overall content of soluble proteins was higher than the control, indicating that soluble protein content increased following infection by the strain, which enhanced the plant's resistance capabilities.