A small assortment of microbial proteins have the ability to activate defense responses and induce necrosis in plant cells through cell signaling pathways. These proteins are of interest because of their potential use as bioherbicides and inducers of plant resistance in agriculture. A 24-kDa protein (Nep1) was purified from culture filtrates of Fusarium oxysporum, and the effects of this protein on weed leaves were investigated. This protein induced necrosis in detached leaves of Papaver somniferum, Lycopersicon esculentum, Malva neglecta, and Acroptilon repens when taken up through the petiole. The pattern and level of necrosis were dependent on the plant species. Treatment with Nep1 induced the production of ethylene in isolated leaves of various species, and the level of ethylene response was shown to be correlated to the concentration of the protein. Pretreating leaves of P. somniferum, L. esculentum, M. neglecta, and Cardaria draba with 100 µl L−1 ethylene enhanced the protein induction of ethylene biosynthesis in those leaves. Application of Nep1 (200 nM) as a spray to intact plants of Abutilon theophrasti, P. somniferum, Centaurea solstitialis, Centaurea maculosa, and Sonchus oleraceus resulted in extensive necrosis of leaves within 48 h. The results of this research are supplemental to our understanding of the role of specific polypeptides in plant/microbe interactions and demonstrates for the first time that a fungal protein can cause extensive necrosis when applied to weed species as a foliar spray.