Curvularia lunata(No. CLST-01), a fungal pathogen isolated from the threeleaf arrowhead (Sagittaria trifoliaL.), has been proposed as a potential mycoherbicide for grass weeds. This paper investigated the physiological and biochemical effects of CLST-01 phytotoxic ethyl acetate fungi extract on the leaves of the threeleaf arrowhead. The results showed that the ethyl acetate fungi extract from CLST-01 can accelerate damage to the cell membrane, increase the production of malondialdehyde, and damage the cellular structure, which could decrease the number of chloroplasts after 96 h treatments. In addition, the content of chlorophyll was reduced by 49.5%, and the net photosynthetic rate, stomatal conductance, and transpiration rate were inhibited. The rates of inhibition were 90.13%, 83.74%, and 79.31%, respectively, and the intercellular CO2concentration increased by 51.87% on Day 9 after treatment with a concentration of 200 μg/mL. In summary, the phytotoxic ethyl acetate fungal extract fromC. lunataCLST-01 can inhibit the photosynthesis of the threeleaf arrowhead leaves, destroy the ultrastructure of leaves, and affect the growth of this invasive weed. Therefore, it has the potential to be developed into a mycoherbicide for weed control in crops as a natural photosynthetic inhibitor.