Tenuazonic acid (TeA), a naturally occurring product of Alternaria alternata, a pathogen to croftonweed, was discovered to be a novel natural photosystem II (PSII) inhibitor. However, herbicidal activity of AAC-toxin, a metabolite of this fungus containing TeA as the main active ingredient, has not been evaluated systematically. In this study, we conducted activity-evaluation experiments in the laboratory, greenhouse, and field trials to assess the herbicidal potential of this fungal metabolite. AAC-toxin had high herbicidal activity on all species tested: croftonweed, large crabgrass, barnyardgrass, redroot pigweed, and eclipta. The AAC-toxin caused brown, leaf spot symptoms and leaf necrosis, subsequently killing the seedlings. When AAC-toxin was applied POST at 83 ml ai/ha, more than 95% of large crabgrass, barnyardgrass, and redroot pigweed plants were controlled 2 d after treatment in field trials. It can be concluded that AAC-toxin has broad-spectrum, rapid, and high herbicidal activity similar to that of paraquat and may have the potential to be developed as a microbe-based herbicide.