Bovine pericardium is the major natural source of patches and aortic valve substitutes in cardiac repair procedures. However, long-term tissue durability and biocompatibility issues lead to degeneration (e.g., calcification) that requires reoperation. Tissue preparation strategies, including glutaraldehyde fixation, are reasons for the deterioration of pericardial tissues. We describe a pretreatment procedure involving sterilization and cross-linking combined with ultraviolet (UV) irradiation and low-energy electron irradiation (SULEEI). This innovative, glutaraldehyde-free protocol improves the mechanical aspects and biocompatibility of porcine pericardium patches. We adopted the SULEEI protocol, which combines decellularization, sterilization, and cross-linking, along with UV irradiation and low-energy electron irradiation, to pretreat bovine pericardium. Biomechanics, such as ultimate tensile strength and elasticity, were investigated by comparing SULEEI-treated tissue with glutaraldehyde-fixed analogues, clinical patch materials, and an aortic valve substitute. Histomorphological and cellular aspects were investigated by histology, DNA content analysis, and degradability. Mechanical parameters, including ultimate tensile strength, elasticity (Young's modulus), and suture retention strength, were similar for SULEEI-treated and clinically applied bovine pericardium. The SULEEI-treated tissues showed well-preserved histoarchitecture that resembled all pericardial tissues investigated. Fiber density did not differ significantly. DNA content after the SULEEI procedure was reduced to less than 10% of the original tissue material, and more than 50% of the SULEEI-treated pericardium was digested by collagenase. The SULEEI procedure represents a new treatment protocol for the preparation of patches and aortic valve prostheses from bovine pericardial tissue. The avoidance of glutaraldehyde fixation may lessen the tissue degeneration processes in cardiac repair patches and valve prostheses.