Eight alkaloids (1–8) were isolated from Ruta graveolens, and their herbicide activities were evaluated through in vitro, semivivo, and in vivo assays. The most relevant results were observed for Compounds 5 and 6–8 at 150 μM, which decreased dry biomass by 20% and 23%, respectively. These are significant results since they presented similar values with the positive control, commercial herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Based on the performed assays, Compound 5 (graveoline) is classified as an electron-transport inhibitor during the light phase of photosynthesis, as well as a plant-growth regulator. On the other hand, Compounds 6–8 inhibited electron and energy transfers, and are also plant-growth inhibitors. These phytotoxic behaviors based on acridone and quinolone alkaloids may serve as a valuable tool in the further development of a new class of herbicides since natural products represent an interesting alternative to replace commercial herbicides, potentially due their low toxicity.