Women’s reproductive health is a severely understudied field and is associated with an abundance of clinically significant pathologies such as pelvic organ prolapse (POP) and preterm birth (PTB). While the exact etiologies of these disorders are unknown, onset is understood to be predominately due to failure of the muscles and connective tissues of the pelvic floor. These failures are attributed to the suboptimal remodeling and maladaptive constituent turnover of the soft tissue extracellular matrix (ECM) including elastic fibers, collagen fibers, glycosaminoglycans / proteoglycans (GAGs/PGs), and smooth muscle cells. Both the vagina and the uterosacral ligament (USL) act as supportive structures that maintain the healthy state of the pelvic floor. Disruption of the elastic fiber and GAG metabolism within the extracellular matrix of these tissues has been suggested to result in the mechanical failure associated with pelvic floor disorders, however, the relationship between microstructure and mechanics is relatively unknown. Histological analysis paired with the biaxial testing of these pelvic floor supporting structures is an important step in providing additional insight into the specific structure-function mechanisms of these tissues. Therefore, the overall objective of this study is to establish a basic understanding of the structure and function of the extracellular matrix constituents within vital soft tissue (vagina, USL) of the murine and human pelvic floor. To elucidate the role of specific constituents, vaginal tissue from female mice in estrus were tested using an extension-inflation biaxial testing protocol pre and post digestion of both elastin and hyaluronan, the most abundant GAG in the extracellular matrix. The digestion of elastin (supported by histological area fraction analysis) resulted in a decreased compliance and an increased dilation of the vaginal wall. Results from the digestion of hyaluronan were inconclusive, as the mechanical response varied with treatment time. Finally, human uterosacral ligaments of postmenopausal, prolapsed patients were tested on a custom built planar biaxial device. This study revealed an inverse relationship between the mechanical stiffness parameters and the ratio of elastin to collagen within the USL. The present findings provide useful information to help understand the etiology behind POP, and help in the development of computational models for the prediction and assessment of observed microstructural changes associated with soft tissue failure.