Bioprosthetic valves created from chemically treated natural tissues such as bovine pericardial biomaterial are used as heart valve scaffolds. Methods currently available for sterilization of biomaterial for transplantation include the application of gamma radiation and chemical sterilants. These techniques, however, can be problematic because they can be expensive and lead to a reduction in tissue integrity. Therefore, improved techniques are needed that are cost effective and do not disrupt the physical properties, functionality, and lifespan of the valvular leaflets. This study examined a novel technique using nonthermal microwave radiation that could lead to the inactivation of bacteria in bovine pericardial biomaterial without compromising valve durability. Two common pathogenic species of bacteria, Escherichia coli and Staphylococcus aureus, were used as test microorganisms. Optimized microwave parameters were used to determine whether inactivation of pathogenic bacteria from bovine pericardium could be achieved. In addition, the effect of microwave sterilization on tissue integrity was examined. The mechanical properties (assessed using dynamic mechanical analysis) and tensile strength testing (using a Universal Tensile Tester) as well as thermal analysis (using thermogravimetric analysis and differential scanning calorimetry) indicated that microwave sterilization did not compromise the functionality of bovine pericardial biomaterial. Scanning electron microscopy imaging and cytotoxicity testing also confirmed that the structure and biocompatibility of transplant biomaterial remained unaltered after the sterilization process. Results from the application of this new microwave (MW) sterilization technique to bovine pericardium showed that near-complete inactivation of the contaminant bacteria was achieved. It is concluded that nonthermal inactivation of pathogenic bacteria from bovine pericardial biomaterial could be achieved using microwave radiation.