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Reactive oxygen species (ROS) play an active role in plant defense. Polyphenol oxidase (PPO) and peroxidase (POD) participate in the synthesis of phytoalexins. The comparative activities of ROS, including hydrogen peroxide (H₂O₂), superoxide anions (O₂⁻), and hydroxyl radicals (·OH), against the fungal pathogen <i>Colletotrichum gloeosporioides</i> from papaya fruit were evaluated. The effects of ROS on PPO and POD activities in papaya fruit inoculated with <i>C. gloeosporioides</i> and the development of natural decay in intact fruit were also investigated. ·OH was the most effective in inhibiting conidial germination and mycelial growth of <i>C. gloeosporioides</i> in vitro. However, 20 or 30 mM H₂O₂ exhibited the best control of the three ROS treatments at ameliorating the disease symptoms associated with the highest levels of PPO and POD activities in papaya fruit. Furthermore, the activities of PPO and POD negatively correlated with the disease index. Overall, H₂O₂ treatments can induce the resistance of papaya fruit against <i>C. gloeosporioides</i> owing to the enhanced activities of PPO and POD. Treatment with 20 mM H₂O₂ also significantly reduced the incidence of decay in intact papaya fruit in semi-commercial experiment, which could make it a potential alternative manner to control postharvest disease in papaya fruit.