Glutaraldehyde cross-linked bioprosthetic heart valves fail within 12–15 years of implantation due to limited durability. Glutaraldehyde does not adequately stabilize extracellular matrix components such as glycosaminoglycans and elastin, and loss of these components could be a major cause of degeneration of valve after implantation. We have shown earlier that neomycin-based cross-linking stabilizes glycosaminoglycans in the tissue but fails to stabilize elastin component. Here, we report a new treatment where neomycin and pentagalloyl glucose (PGG) were incorporated into glutaraldehyde cross-linking neomycin-PGG-Glutaraldehyde (NPG) to stabilize both glycosaminoglycans and elastin in porcine aortic valves. In vitro studies demonstrated a marked increase in extracellular matrix stability against enzymatic degradation after cross-linking and 10 month storage in NPG group when compared to glutaraldehyde controls. Tensile properties showed increased lower elastic modulus in both radial and circumferential directions in NPG group as compared to glutaraldehyde, probably due to increased elastin stabilization with no changes in upper elastic modulus and extensibility. The enhanced extracellular matrix stability was further maintained in NPG-treated tissues after rat subdermal implantation for three weeks. NPG group also showed reduced calcification when compared to glutaraldehyde controls. We conclude that NPG cross-linking would be an excellent alternative to glutaraldehyde cross-linking of bioprosthetic heart valves to improve its durability.