Euphorbia heterophylla is a widely distributed weed whose seeds can remain viable in the soil for years, competing with crops. Therefore, natural herbal preparations could be a solution for its more diversified management. This study investigates the efficacy and mode of action of aqueous mixtures of Urochloa ruziziensis stems and Sorghum bicolor roots and stems on E. heterophylla seed germination, seedling development, antioxidant enzyme activity, and respiration. Aqueous mixtures with concentrations of 0 (control), 250, 500, 750 and 1000 ppm were prepared. E. heterophylla seeds were treated with the mixtures and incubated under controlled conditions. Seedling development, respiration, and enzyme activity were assessed after 4 days of incubation and germination was analyzed after 16 days. Urochloa ruziziensis and S. bicolor mixtures presented allelopathic effects on E. heterophylla inhibiting root growth, root fresh and dry weights and induced mitochondrial alterations resulting in oxidative stress, increasing the antioxidant enzymes catalase and peroxidase. U. ruziziensis and S. bicolor aqueous mixtures were found to have potential in controlling the weed E. heterophylla in laboratory tests.