Ovarian cancer (OC) is one of the most common types of cancer among women worldwide. The majority of patients with OC respond to current chemotherapy approaches initially; however, patients are likely to experience cancer recurrence and become resistant to the chemotherapy. Therefore, novel agents for the treatment of OC are urgently required. Chaetocin, a natural product isolated from Chaetomium fungi, has been reported to exhibit anticancer activity against various types of cancer; however, the pharmacological action and detailed mechanism underlying the effects of chaetocin on OC cells remain unclear. Therefore, the present study investigated the cytotoxic effects of chaetocin on OC cells. A Cell Counting kit-8 assay was used to study cell viability, a colony formation assay was used to assess cell proliferation, flow cytometry was used to detect apoptosis, cell cycle and reactive oxygen species (ROS) generation, and western blotting was used to determine the protein levels of poly (ADP-ribose) polymerase, caspase-3 and cleaved-caspase-3. The results demonstrated that chaetocin significantly decreased the viability of OC cells. Chaetocin inhibited the proliferation and induced G2/M phase arrest of the OVCAR-3 OC cell line. Additionally, chaetocin induced apoptotic cell death in OVCAR-3 cells via the caspase pathway. It was observed that chaetocin induced the accumulation of ROS in OVCAR-3 cells. Treatment with the ROS scavenger N-acetyl-L-cysteine reversed the apoptotic effects and activation of the caspase pathway induced by chaetocin. Collectively, these results revealed that chaetocin suppressed the proliferation and promoted the caspase-dependent apoptosis of OC cells by increasing the levels of ROS. Therefore, chaetocin may serve as a potential therapeutic agent for the treatment of OC.