Trabeculae carneae account for a significant portion of human ventricular mass, despite being considered embryologic remnants. Recent studies have found trabeculae hypertrophy and fibrosis in hypertrophied left ventricles with various pathological conditions. The objective of this study was to investigate the passive mechanical properties and microstructural characteristics of trabeculae carneae and papillary muscles compared to the myocardium in human hearts. Uniaxial tensile tests were performed on samples of trabeculae carneae and myocardium strips, while biaxial tensile tests were performed on samples of papillary muscles and myocardium sheets. The experimental data were fitted with a Fung-type strain energy function and material coefficients were determined. The secant moduli at given diastolic stress and strain levels were determined and compared among the tissues. Following the mechanical testing, histology examinations were performed to investigate the microstructural characteristics of the tissues. Our results demonstrated that the trabeculae carneae were significantly stiffer (Secant modulus SM2 = 80.06 ± 10.04 KPa) and had higher collagen content (16.10 ± 3.80%) than the myocardium (SM2 = 55.14 ± 20.49 KPa, collagen content = 10.06 ± 4.15%) in the left ventricle. The results of this study improve our understanding of the contribution of trabeculae carneae to left ventricular compliance and will be useful for building accurate computational models of the human heart.