Malignant ascites in hepatocellular carcinoma is usually a sign of advanced disease and poor prognosis and is also thought to be associated with chronic inflammation mediated by neutrophil extracellular trap (NET) networks. Although ozone, a strong oxidant, has significant antibacterial and anti-inflammatory effects, its effectiveness in treating malignant liver ascites is unclear. We first measured the levels of NETs in the peripheral blood of patients with liver cancer and healthy individuals. Next, we constructed the H22 tumor-bearing mouse model and observed the abdominal girth, body weight, survival rate, and survival time in each group; we marked the proteins associated with NETs in mouse intestinal tissues by immunofluorescence; cf-DNA and cytokines in ascites such as: tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), matrix metalloprotein 9 (MMP-9), and interferon gamma (IFN-γ) levels in ascites were measured by enzyme-linked immunosorbent assay. The expression levels of phosphorylated adenylate-activated protein kinase (P-AMPK) and scavenger receptor-A (SR-A) were detected by immunocytochemistry in the intestinal tissues of each group of mice. We further examined the expression of P-AMPK and SR-A proteins in ascites deposits by Western blotting. The results show, the plasma levels of NETs were higher in patients with hepatocellular carcinoma than in normal subjects (P < 0.01). Abdominal girth and body weight were significantly reduced in the ozone-treated group compared with the model group, while survival and survival time were dose dependently increased (both P < 0.05). NET-associated guanine histone H3 and myeloperoxidase were abundantly expressed at neutrophil aggregates in the intestinal tissues of the model mice, whereas their expression was significantly reduced in the ozone-treated group. The levels of cf-DNA, IL-6, IFN-γ, MMP-9, VEGF, and TNF-α were dose dependently increased in the ascites of H22 tumor-bearing mice in the ozone-treated group compared with the model group (all P < 0.01), while the expression of P-AMPK and SR-A proteins was increased in the ozone-treated group compared with the model group. Ozone showed significant antiperitoneal fluid production properties in H22 tumor-bearing mice, and ozone reduced peritoneal fluid production by activating AMPK and up-regulating SR-A phagocytosis damage-associated molecular patterns to reduce the production of NETs. This suggests that ozone could be used as a new drug for the treatment of malignant ascites in hepatocellular carcinoma.