The presence or absence of light is one of the most significant environmental factors affecting plant growth and defence. Therefore, the selection of the most appropriate time of application may maximize the benefits of photosynthetic inhibitors. In this work, the concentration and daytime or night-time-dependent effects of bentazon were tested in soybean and common ragweed. The recommended dose (1440 g ha−1) and also half the recommended dose significantly reduced the maximum quantum yield (Fv/Fm) and increased H2O2 levels in common ragweed. Interestingly, bentazon did not change Fv/Fm in soybean. The activity of superoxide dismutase changed in a dose-dependent manner only in common ragweed. The activity of ascorbate peroxidase, catalase and glutathione S-transferase (GST), as well as the contents of ascorbate (AsA) and glutathione (GSH) did not change significantly in this plant species. In soybean, alterations in H2O2 levels were lower but GST and APX activity, as well as AsA and GSH levels were higher compared to common ragweed. At the same time, the rate of lipid peroxidation and ion leakage increased upon bentazon, and were higher in the light phase-treated leaves in the case of both plant species. These results can contribute to optimizing the effects and uses of herbicides in agriculture.