Inhibitory effects of two sesquiterpene lactones, costunolide and parthenolide, isolated from dichloromethane extract of the stem bark of southern magnolia and a parthenolide derivative, 1,10-epoxyparthenolide, were evaluated on germination and seedling growth of wild oat. The sesquiterpene lactones effected a significant reduction of seed germination, particularly at the highest concentrations of 200, 400, and 600 mg L−1, with costunolide being the most active one. Furthermore, the three sesquiterpenes strongly inhibited root and shoot growth of the weed. However, the inhibition of root growth by all compounds was greater than that of shoot growth. Parthenolide inhibited growth of both root and shoot more strongly than the other compounds and a reference herbicide imazamethabenz. At a concentration of 100 mg L−1, parthenolide caused 87 and 41% growth inhibition of root and shoot, respectively. Parthenolide was tested for its effect on acetolactate synthase (ALS) activity. The compound inhibited the enzyme in a concentration-dependent manner, with 50% inhibition of 51.44 µM. The results of this study indicated that the herbicidal activity of the isolated sesquiterpene may be attributed to inhibition of ALS. The promising phytotoxic activity of sesquitepene lactones reported here could be considered a starting point for developing environmentally safer herbicides.