Three ethylene diurea (EDU) concentrations (0, 150 and 300 mg/L) were used to evaluate the negative impact of ozone (O3) on two cultivars of Trifolium repens L. Cv. Vardan and Bundel grown under natural field conditions in a suburban area of Varanasi, India. Mean O3 concentrations varied from 30.3 to 46.6 μtg/L during the experimental period. Higher photosynthetic pigments and ascorbic acid concentrations were noticed in both EDU-treated cultivars over non-EDU-treated ones, but a reverse trend was found for lipid peroxidation. Growth parameters and biomass also showed increments under EDU treatment of both cultivars. The ratio of variable fluorescence to maximum fluorescence increased significantly in Vardan but not in Bundel upon EDU treatment. Results revealed that EDU concentration of 300 mg/L was more effective to combat the oxidative stress as well as protecting plants from O3 injury symptoms. The test cultivar Vardan is relatively sensitive to O3, thereby can be used as a bioindicator of O3 pollution in areas having higher O3 concentrations. Results also indicated that Bundel has more efficient antioxidant defense system than Vardan and hence was more tolerant to O3 stress.