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Fresh <i>Codonopsis pilosula</i> is highly susceptible to fungus contamination during post-harvest storage, which not only compromises the quality of <i>C. pilosula</i> but also contributes mycotoxin contamination, posing a significant threat to human health. Studies have indicated that ozone treatment can inhibit post-harvest diseases in fruits and vegetables. The impact of ozone treatment on the disease incidence, disease severity index, and weight loss rate of the fresh <i>C. pilosula</i> infected with <i>Actinomucor elegans</i> was investigated through the spray inoculation of <i>A. elegans</i> on <i>C. pilosula</i> tissues. Changes in the main active ingredients of <i>C. pilosula</i> after ozone treatment were analyzed, and the effects of ozone treatment on the integrity of cell membranes in <i>C. pilosula</i> tissue and reactive oxygen species (ROS) metabolism were studied. The results showed that ozone treatment had a significant inhibitory effect on the <i>A. elegans</i>-induced mucor rot in <i>C. pilosula</i>, significantly reducing the incidence of the disease. Compared with the control group, the ozone-treated group maintained the effective components of <i>C. pilosula</i> well. Furthermore, ozone treatment reduced the cell membrane permeability and Malondialdehyde (MDA) content in <i>C. pilosula,</i> significantly increased the activity of antioxidant enzymes in the ROS metabolism pathway, prevented oxidative stress caused by the accumulation of ROS in <i>C. pilosula</i> tissues, and maintained the integrity of cell membranes.