Tissue-engineered airways have achieved clinical success, but concerns remain about short-term loss of biomechanical properties, necessitating a stent. This study investigated the effect of chemical-enzymatic decellularization on biochemical properties of trachea important for cell attachment and vascularization (fibronectin and laminin) and cartilage matrix homeostasis (type II collagen and glycosaminoglycans (GAG)), as well as biomechanical status. Native trachea was used as a control, and NDC trachea stored in phosphate buffered saline (PBS) in parallel to decellularization was used as a time-matched control. Decellularization removed most cells, but chondrocytes and DNA remained after 25 cycles. Fibronectin was retained throughout the lamina propria and laminin at basement membranes. DNA accumulation along ECM fibres was seen. A decline in soluble collagen was observed in decellularized tissue. GAG content of cartilage rings was reduced, even in PBS control tissue from 20 cycles onwards (p<0.05), but decellularization caused the greatest loss (p<0.01). Tensile strength declined throughout the process, but was significant only at later time points. The data demonstrate that the substantial reduction in GAG might contribute to loss of mechanical integrity of biotracheas. Overcoming structural changes that cause an imbalance in cartilage matrix equilibrium will be necessary to optimize clinical benefit, enabling widespread use of biotracheas.