As a common cause of infertility, pelvic inflammatory disease (PID) is characterized by chronic pain, ectopic pregnancy as well as inflammation and infection of the female upper genital tract. Ozone water, also known as O_3, has been previously reported to be a distinctly effective agent in treating inflammation. During the present study, we asserted the hypothesis that O₃ could be applied by pelvic inflammation and works to regulate the expression of inflammatory factors including interleukin-6 (IL-6), IL-2 and tumor necrosis factor-α (TNF-α). In an attempt to evaluate the effect of O₃ on PID, an acute PID rat model was subsequently established. O₃ at concentrations of 45 μg/mL and 60 μg/mL in addition to levofloxacin (LVLX) was injected respectively into the PID rats in a bid to alter the contents of inflammatory factors and immunologic markers. Hematoxylin-eosin (HE) staining was applied to analyze endometrial inflammation. Reductions to the contents of IL-6 and TNF-α were recorded, while that of IL-2, IgA, IgG, IgM, C3 and C4, and E rosette formation rate and transformation rate of T lymphocytes exhibited notably elevated levels after the PID rats had been injected with 45 μg/mL O₃, 60 μg/mL O₃ or LVLX. The pathological condition of the endometrium in rats with PID was alleviated among the PID rats after injected with the 45 μg/mL O₃, 60 μg/mL O₃ or LVLX. Taken together, the key findings of the current study present evidence demonstrating that the administration of O₃ to the pelvic cavity ameliorated the PID conditions among rat models via inhibition of the necrosis of the endometrial epithelial cells as well as alleviated the inflammatory reactions, highlighting a potential novel PID treatment target.