Currently, no effective crosslinking reagents are available to treat xenogenic decellularized heart valve matrices. The study aim was to evaluate the crosslinking effect of quercetin, catechin, caffeic acid and tannic acid on porcine aortic valve matrices. Cytotoxicity of the different crosslinkers was evaluated. The mechanical properties of crosslinked porcine matrices and control matrices (non-fixed) were examined by tensile strength testing, as was the cytocompatibility of the fixed matrices. Crosslinked and control matrices were implanted subcutaneously in Wistar rats (n = 9) and, after two weeks, their calcium contents were determined using inductively coupled plasma-mass spectrometry. The antibody reaction against porcine tissue in rat serum was also determined. Cytotoxicity studies showed that crosslinkers, even at high concentrations, did not inhibit cell viability. All crosslinkers except tannic acid improved the mechanical strength of acellular porcine matrices. Moreover, the tensile strength of quercetin-fixed matrices was comparable with that of glutaraldehyde (GTA)-fixed leaflets. Light microscopic evaluation showed that crosslinked matrices caused only a mild lymphocytic inflammatory reaction. Furthermore, quercetin-fixed leaflets exhibited a well-preserved matrix without infiltration of CD3+ cells. After two weeks, calcium levels were 206.33 µg/mg for controls (non-fixed), and 151.33 µg/mg, 181 µg/mg and 163.66 µg/mg for quercetin-, catechin-, and caffeic acid-fixed matrices, respectively. At two weeks after implantation the quercetin-crosslinked matrices also elicited the lowest levels of IgG antibodies. The study results identified quercetin as the most suitable crosslinker for heart valve tissue engineering, and a possible alternative to GTA. Further studies are essential to determine whether quercetin crosslinking will allow autologous cell repopulation in order to create a viable heart valve.