Oxidative stress has been shown to be the main cause of protein denaturation and cell death. However, the exact mechanism of antioxidants against oxidative stress-mediated protein damage and cell cytotoxicity remains largely unknown. Therefore, in this paper, the protective effects of acteoside against H2O2-inudced hemoglobin structural changes and cardiomyocyte toxicity as models were determined by different techniques. Fluorescence quenching as well as molecular docking simulation studies showed that acteoside potentially interacted with hemoglobin in a one: one binding mode mediated mainly by the involvement of hydrophobic forces. It was also shown that H2O2 caused significant changes in the structure of hemoglobin as well as heme degradation, reversed by acteoside. Additionally, UV-visible studies exhibited that acteoside decreased the generation of methemoglobin triggered by H2O2. Cellular assays displayed that acteoside could mitigate the cardiomyocyte toxicity induced by H2O2 through regulation of LDH release, generation of reactive oxygen species (ROS) and 3,4-methylenedioxyamphetamine (MDA), mitochondrial membrane potential (MMP) collapse, as well as superoxide dismutase (SOD), catalase (CAT), caspase-9, and caspase-3 activities. In conclusion, acteoside may hold great promise for the control of protein-related disorders as well as cardiovascular diseases.