This study evaluates the effects of two decellularization protocols, enzyme-detergent (ED) and detergent-detergent (DD), on the structural and biomechanical properties of human urethral tissue. Urethral samples from 18 individuals were divided into ED ( = 7) and DD ( = 11) groups, with native samples ( = 3) serving as controls. Histological and ultrastructural analyses confirmed that both protocols effectively removed cellular content while preserving essential extracellular matrix (ECM) elements, such as collagen and elastic fibers. Immunohistochemical staining for collagen IV and fibronectin revealed no significant differences between decellularized and native tissues, indicating intact ECM structure. Biomechanical testing demonstrated that DD-treated tissues had significantly lower Cauchy stress (1494.8 ± 518.4 kPa) when compared to native tissues (2439.7 ± 578.7 kPa, = 0.013), while ED-treated tissues were similar to both groups. Both decellularized groups exhibited reduced stretch at failure and elastic modulus compared to native tissues. Cytotoxicity assays using adipose-derived stem cells demonstrated no signs of toxicity in either protocol. Overall, both ED and DD protocols effectively preserved the urethral ECM structure and mechanical properties, making them suitable for potential use in tissue-engineered grafts and for biobanking purposes. Further research is needed to refine and optimize decellularization methods to improve scaffold recellularization and ensure clinical safety and efficacy.