Following prometryne treatment, Polygala tenuifolia Willd. (Polygala) seedlings were investigated histochemically and biochemically to determine the effects of prometryne on root activity, lipid peroxidation, membrane integrity, reactive oxygen species (ROS) accumulation and antioxidant enzyme activity. There was no significant difference between untreated controls and seedlings treated with 10 mg/L prometryne in terms of root activity, membrane permeability and ROS production. Seedlings treated with 25-400 mg/L prometryne showed significantly accelerated ROS production, enhanced loss of membrane integrity and inhibited root cell viability. Histochemical staining with Schiff's reagent, Evans blue, nitroblue tetrazolium and 3,3-diaminobenzidine was positively correlated with the accumulation of malondialdehyde (MDA), loss of membrane integrity, generation of superoxide radicals (O₂^{· -}) and generation of hydrogen peroxide (H₂O₂), respectively. Prometryne-induced oxidative stress triggered significant changes in the antioxidant defense machinery. At lower prometryne concentrations, the antioxidant defense system of Polygala seedlings demonstrated very efficient and coordinated enzymatic (superoxide dismutase; catalase; ascorbate peroxidase; peroxidase) and non-enzymatic (non-protein amino acids; proline) scavenging of ROS to regulate the cascades of uncontrolled oxidation and protect plant cells from oxidative damage. Higher prometryne levels increase oxidative stress through excessive production of ROS and destruction of antioxidant systems, ultimately leading to seriously damaged Polygala seedlings. The presented results indicate that Polygala seedlings are very sensitive to prometryne exposure during germination; however, the application of 10 mg/L is safe for seedling growth. Treatment at 25 mg/L prometryne can cause significant oxidative stress and lipid peroxidation as well as damage cell membrane integrity, leading to decreased root activity and inhibited seedling growth. These results may provide a theoretical basis for the mechanisms of herbicide resistance and the safe use of herbicides during Polygala cultivation.