Abstract Knowing the mechanical properties of cardiac myofibrils isolated from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can provide valuable insight into the structure and function of the heart muscle. Previous studies focused mostly on studying myofibrillar stiffness using simplified elastic models. In this study, the mechanical properties of myofibrils isolated from hiPSC-CMs were measured using atomic force microscopy (AFM). The quasi-linear viscoelastic (QLV) model was used to interpret the elastic and viscous properties of myofibrils. Since there have been no previous studies on the viscoelastic properties of myofibrils extracted from hiPSC-CMs, myofibrils extracted from porcine left-ventricular (LV) tissue were used to compare and verify experimental processes and QLV model parameters. The elastic modulus of myofibrils extracted from porcine LV tissue was determined to be 8.82 ± 6.09 kPa which is consistent with previous studies which reported that porcine LV tissue is less stiff on average than mouse and rat cardiac myofibrils. The elastic modulus of myofibrils extracted from hiPSC-CMs was found to be 9.78 ± 5.80 kPa, which is consistent with the range of 5–20 kPa reported for myofibrils extracted from the adult human heart. We found that myofibrils isolated from hiPSC-CMs relax slower than myofibrils extracted from porcine LV tissue, particularly in the first 0.25 s after the peak stress in the stress relaxation test. These findings provide important insights into the mechanical behavior of hiPSC-CMs and have implications for the development of treatments for heart diseases.