Our objective was to study the effect of glutaraldehyde (GLU) concentration, heat, and photooxidation on mechanical properties and calcification of bovine pericardium grafts in an in vivo model. Fresh pericardia were treated as follows: 0.625% GLU for 7 days (standard); 0.625%, 1%, and 3% GLU at 4°C for 20 days and 50°C for additional 20 days; irradiation in cross-linking medium with metilene blue at 0°C for 8 hours. Tissues were subjected to tensile mechanical tests (n = 76). Fixed patches were subcutaneously implanted in mice for 50 days (n = 16 per treatment). Calcification was assessed by atomic absorption spectrophotometry (n = 55) and von Kossa staining (n = 28). Analysis of variance and Tukey's test were used for statistical analysis. The 3% GLU and 3% GLU + heat treatments showed an enhancement of the mechanical properties above standard treatment. No significant difference was found in calcification between treatments. The 3% GLU treatment enhances the mechanical properties of the tissue above standard treatment without increasing calcification and without applying heat; therefore it is recommended for high-strength applications. Supplementary treatments to decrease calcification could be combined with this methodology to obtain a high-strength–low-calcification biomaterial for manufacturing of long-term cardiovascular grafts.