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Tanneries are increasingly focusing on environmentally friendly processes, emphasizing the concept of clean production to minimize the environmental impact. This study investigated the eco-friendly application of an α-amylase/neutral protease system (ANS) in a single-step process for unhairing, fiber opening, and bating, providing an effective alternative to the conventional complex chemical-based lime-sulfide system (LSS). A mathematical model was established using response surface methodology (RSM) to optimize the operational conditions, yielding a 0.3 wt% α-amylase/0.5 wt% neutral protease solution at 28.4 °C for 16.6 h. The effects on unhairing and fiber opening were evaluated using scanning electron microscopy (SEM), while the effect on bating was accessed by the removal of carbohydrates and proteoglycans. The obtained leathers had comparable physical properties to their conventional counterparts, with enhanced hydrothermal shrinkage temperature and softness. In terms of environmental benefits, the optimized ANS could significantly decrease more than 90% of the chemical oxygen demand (COD), NH3–N, and Cl−, respectively, along with a 73.91% reduction in total solids (TS). Cost analysis indicated direct savings with a significant cost reduction of 30.98% of the ANS compared to that of the LSS, as well as additional indirect cost reduction through improved production efficiency and streamlined wastewater treatment processes. The use of electricity and water was reduced by 44.6% and 93.9%, respectively, which could reduce greenhouse gas emissions by 44.6%. This study highlighted the potential of α-amylase/neutral protease as a sustainable and cost-effective alternative in leather manufacturing that aligns with environmentally friendly practices. [ABSTRACT FROM AUTHOR]