Scaffolds used in tissue engineering can be obtained from synthetic or natural materials, always focusing the effort on mimicking the extracellular matrix of human native tissue. In this study, a decellularization process is used to obtain an acellular, biocompatible non-cytotoxic human pericardium graft as a bio-substitute. An enzymatic and hypertonic method was used to decellularize the pericardium. Histological analyses were performed to determine the absence of cells and ensure the integrity of the extracellular matrix (ECM). In order to measure the effect of the decellularization process on the tissue's biological and mechanical properties, residual genetic content and ECM biomolecules (collagen, elastin, and glycosaminoglycan) were quantified and the tissue's tensile strength was tested. Preservation of the biomolecules, a residual genetic content below 50 ng/mg dry tissue, and maintenance of the histological structure provided evidence for the efficacy of the decellularization process, while preserving the ECM. Moreover, the acellular tissue retains its mechanical properties, as shown by the biomechanical tests. Our group has shown that the acellular pericardial matrix obtained through the super-fast decellularization protocol developed recently retains the desired biomechanical and structural properties, suggesting that it is suitable for a broad range of clinical indications.