Photooxidation is a method of tissue fixation resulting in protein crosslinking due to illumination in the presence of a dye. The aim of the study was to evaluate the impact of dyes, photooxidation time and the type of applied light on the porcine pericardial collagen crosslinking. The collagen modifications were evaluated on the basis of pericardial sensitivity to pepsin digestion. The hydrolysate components were evaluated qualitatively and quantitatively. All hydrolysates contained collagen alpha chains, their aggregates and degradation products. Methylene blue and methylene green-mediated 4 h photooxidation in the presence of visible light caused similar decrease in pericardium sensitivity to pepsin. However, both fixation types generated remarkable amounts of alpha chain degradation products. The prolongation of photooxidation time to 8 h did not increase the pericardial sample resistance to pepsin. Moreover, these sample hydrolysates revealed an elevated alpha chain content. Violet light mediated photooxidation did not alter pericardial sensitivity to pepsin when compared with fixation under visible light. Nevertheless, violet light fixed tissues displayed a decrease in collagen degradation products. The application of violet light in photooxidation of porcine pericardium will probably allow to obtain enzyme resistant bioprostheses with better mechanical properties compared with those obtained after visible light mediated process.