This study investigated the calcification process that occurred on chemically treated bovine pericardium substrata through tests with simulated body fluid solutions. The use of bovine pericardium bioprosthetic valves in heart valve surgery has a significant drawback due to the calcification processes. Thus, many routes such as chemical treatments in the substratum or the adoption of systemic therapies are considered in the literature with the intention to inhibit or to decelerate this process. The presented treatment using the two different phenetylamine-diepoxide solutions showed no effects on calcification experiments as showed by the tests. However, the lyophilized bovine pericardium samples, treated with both solutions, did not show any detectable phosphate deposits. The lyophilization of bovine pericardium before chemical treatments with cross-link agents as epoxy compounds may be an alternative to the conventional calcification prevention methods, but further investigations are recommended to check if the same behavior is found in all lyophilized systems.