The atrioventricular heart valve (AHV) leaflets have a complex microstructure composed of four distinct layers: atrialis, ventricularis, fibrosa and spongiosa. Specifically, the spongiosa layer is primarily proteoglycans and glycosaminoglycans (GAGs). Quantification of the GAGs mechanical contribution to the overall leaflet function has been of recent focus for aortic valve leaflets, but this characterization has not been reported for the AHV leaflets. This study seeks to expand current GAG literature through novel mechanical characterizations of GAGs in AHV leaflets. For this characterization, mitral and tricuspid valve anterior leaflets (MVAL and TVAL, respectively) were: (i) tested by biaxial mechanical loading at varying loading ratios and by stress-relaxation procedures, (ii) enzymatically treated for removal of the GAGs and (iii) biaxially mechanically tested again under the same protocols as in step (i). Removal of the GAG contents from the leaflet was conducted using a 100 min enzyme treatment to achieve approximate 74.87% and 61.24% reductions of all GAGs from the MVAL and TVAL, respectively. Our main findings demonstrated that biaxial mechanical testing yielded a statistically significant difference in tissue extensibility after GAG removal and that stress-relaxation testing revealed a statistically significant smaller stress decay of the enzyme-treated tissue than untreated tissues. These novel findings illustrate the importance of GAGs in AHV leaflet behaviour, which can be employed to better inform heart valve therapeutics and computational models.