Background: This research aimed to investigate the cytotoxicity of methanol extract of Smallanthus sonchifolius leaf (YLE) against a human hepatocellular carcinoma cell line (HepG2). This plant is currently used as a traditional herbal remedy in the treatment of liver diseases in some rural parts of Myanmar. Methods: The cytotoxic activity of the plant extract against the cancerous cell line was assessed using an MTT assay. YLE demonstrated a significant effect (IC50 = 58.2 ± 1.9 μg/mL) on anti-cancer activity, which was further investigated using various assays including an in vitro cell migration assay, a colony formation assay, cell cycle analysis, western blot analysis, and a ROS assay. The significance of the phytochemical constituents of YLE could be identified using LC/Q-TOF-MS techniques. Results: We putatively identified the active components in YLE, which were possibly melampolide-type sesquiterpenoids. YLE showed an inhibitory effect on HepG2 cell proliferation and cell migration. YLE also induced cell cycle arrest and necrosis in a dose-dependent manner. Additionally, YLE significantly suppressed ROS formation in HepG2 cells. Conclusions: These findings suggest that YLE is sufficient for application as a promising anti-liver drug in herbal medicine.