Updated project metadata. Structural cardiac lesions are often surgically repaired using prosthetic patches, which can be of biological or synthetic nature. In the current clinical scenario, amongst biological patches, are gaining very much interest those derived from decellularization of xenogeneic scaffold. These patches maintain their natural architecture of the extracellular matrix (ECM) after the removal of their native cells. Moreover, once implanted in the host, they promote tissue regeneration and repair, encourage angiogenesis, migration, proliferation, and host’s cell differentiation mechanisms. Finally, in the host, decellularized xenogeneic patches, after cells repopulation, reduce immuno-mediated response against the graft thus preventing device failure. Small intestinal submucosa (SIS) from porcine showed such properties in alternative clinical scenarios. The US FDA approved its use in humans for urogenital procedures, such as hernia repair, cystoplasties, ureteral reconstructions, stress incontinence, Peyronie’s disease, penile chordee, and urethral reconstruction for hypospadias and strictures. In addition, it has also been successfully used for the skeletal muscle tissue reconstruction in young patients. However, for cardiovascular applications, the results are still controversial. In this study, we aimed to validate our decellularization protocols for SIS, which is based on the use of Tergitol 15 S 9, by comparing it to our previous and efficient method (Triton X 100) which is not more available on the market. For both treatments, we evaluated the preservation of the ECM ultrastructure, mechanics, biocompatibility and final bioinductive capabilities. The overall analysis shows that the SIS tissue is macroscopically distinguishable into two regions, smooth or wrinkle, which are equivalent for ultrastructure and biochemical profile. Furthermore, Tergitol 15 S 9 treatment do not modify the mechanics of the tissue, resulting comparable to the native one and confirming the superior preservation of the collagen fibers. In summary, our study showed that the SIS decellularized with Tergitol 15 S 9 guarantees higher performance compared to Triton X 100 in all the fields of characterization explored and for all the components of the SIS: wrinkled and smooth.