Ovarian cancer is one of the leading causes of cancer-related death among women worldwide and accounts for 4% of all cancer cases in female patients. To date, ovarian cancer has the poorest prognosis among all types of gynecological cancer; thus, it is necessary to identify prospective therapeutic options. Previous studies have demonstrated the involvement of reactive oxygen species (ROS) in the cytotoxicity of various anticancer drugs against several types of carcinoma, including ovarian cancer. The present study aimed to investigate the anticancer effects of Siomycin A, a thiopeptide antibiotic, on the ovarian cancer cell lines PA1 and OVCAR3. To determine the viability of these cells following exposure to Siomycin A, the MTT assay was used, and apoptosis was determined by ELISA. In addition, mitochondrial membrane potential was determined by JC1 staining, and cellular ROS levels were assessed by dichlorodihydrofluorescein diacetate staining in the presence and absence of antioxidant NAC. The subsequent levels of antioxidant enzymes and glutathione were also determined following Siomycin A treatment in the two cell lines. A combination study with Siomycin A and cisplatin indicated enhanced efficiency of the drugs on ovarian cancer cell viability. The results of the present study also demonstrated that Siomycin A induced ROS production, inhibited the major antioxidant enzymes, including catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and intracellular GSH in PA1 and OVCAR3 cells, and inhibited the cell viability with an IC50 of ~5.0 and 2.5 µM after 72 h respectively compared with the untreated controls. Additionally, the Siomycin A-induced ROS production further targeted apoptotic cell death by impairing the mitochondrial membrane potential and modulating the levels of pro- and antiapoptotic proteins compared with those in the corresponding control groups. The administration of the antioxidant N-acetylcysteine significantly abrogated the cytotoxic effects of Siomycin A. In conclusion, the results of the present study demonstrated the role of ROS in Siomycin A-mediated cytotoxicity in ovarian cancer cells.