Glutaraldehyde (GA)-treated bovine pericardium is still the gold standard for the fabrication of bioprostheses needed for the surgical treatment of valvular malfunction. Although excellent stability and low immunogenicity are accomplished, the application of GA is considered to be causal for structural valve deterioration, diminishing the long-term durability of bioprosthetic tissue. The novel GA-free SULEEI-treatment of bovine pericardium combines decellularization, riboflavin/UVA-cross-linking, and low-energy electron beam irradiation. In the present study, we initiated an in vivo application. We used a subcutaneous rat model to compare the immune and tissue responses, calcification propensity, and biomechanical properties of the alternatively prepared SULEEI bovine pericardial tissue with standard glutaraldehyde-fixed and industrially produced bovine pericardial patch material. SULEEI pericardium evokes a similar immune reaction and tissue response as the control standard bovine patch material. The calcification propensity of SULEEI tissue was low, and biomechanical analysis revealed a heterogeneous but similar pattern in tissue stiffness compared to the control patch. The results of this study highlight the potential of SULEEI-treated bovine pericardial tissue as a candidate for cutting-edge cardiovascular and valvular biomaterials in reconstructive surgery.