Oxidative stress significantly contributes to ageing and disease, with antioxidants holding promise in mitigating its effects. Functional foods rich in flavonoids offer a potential strategy to mitigate oxidative damage by free radicals. We investigated the protective effects of mulberry leaf flavonoids (MLF) against HO-induced oxidative damage in HepG2 cells. It assessed the inhibitory effect of MLF (62.5-500 μg/mL) on HO-induced oxidative damage by analyzing cellular morphology and oxidative stress markers, including ROS production, mitochondrial membrane potential, antioxidant enzyme levels, MDA, and apoptosis-related proteins. The results demonstrated that MLF prevented spiny cell formation triggered by 750 μM HO and significantly reduced ROS levels, restored mitochondrial membrane potential, decreased lactate dehydrogenase and alanine transaminase leakage, and reduced MDA content induced by HO. MLF also modulated antioxidant enzymes and attenuated oxidative damage to HepG2 cell DNA, as confirmed by staining techniques. These findings indicate the potential of MLF as a hepatoprotective agent against oxidative damage in HepG2 cells.