Collagen is a widely used raw material for biomaterial manufacture, which generally depends on chemical modifications of this fibrillar protein with cross-linking agents to improve biocompatibility and mechanical properties. However, cross-linking reduces the natural properties of collagen, such as low immune response, low toxicity as well as the ability to promote cellular growth and attachment. In this work, the modifications promoted by 1,4-dioxane solvent on the collagen present in native bovine pericardium (NBP) matrix routinely used in bioprosthesis manufacture, with or without subsequent cross-linking by glutaraldehyde, has been studied. The structural changes of NBP evaluated by scanning electron microscopy show that 1,4-dioxane induces a more homogeneous material by increasing aggregation of collagen fibers, while transmission scanning electron microscopy shows that natural collagen fibril arrangement, integrity, and the D-periodicity pattern are maintained by solvent treatments. Measurements of thermal stability and resistance to collagenase enzymatic digestion of NBP matrices treated with 1,4-dioxane show an increase in melting temperature and decrease in biodegradability, as compared to native pericardium. Cross-linking with glutaraldehyde improves all the analyzed NBP properties, which are not impaired by previous treatment with 1,4-dioxane. Histological evaluation of NBP submitted to 1,4-dioxane treatment shows lower lipid and cell contents and improvement in other morphologic characteristics compared to native pericardium. Altogether, these results suggest the use of 1,4-dioxane organic solvent as an alternative non-cross-linking treatment for direct utilization on rich collagen matrices, resulting in materials with improved biocompatibility and physicochemical properties suitable for tissue engineering.