In this work is described the isolation of a new proteases-producing strain of Bacillus subtilis, screened from aerobic tannery sludge, to be applied in leather production. The optimization of culture conditions to enhance the proteolytic activity was carried out using central composite design. The enzymatic extract was characterized and the hide unhairing and the inter-fibrillary removal capabilities of the enzymatic extract were evaluated by scanning electron microscopy and by the determination of proteoglycans and glycosaminoglycans. The leather quality obtained with this enzymatic preparation was assessed for possible damages to hide collagen by measuring the amount of hydroxyproline released into the reaction medium. Temperature was the most significant factor for culture conditions optimization. The crude enzymatic extract showed the best values for proteolytic activities at pH 9 and 10, temperature between 37 and 55 °C, and showed good thermal stability up to 45 °C. The treated hides presented few remaining hairs; for the enzymatic process, the removal of inter-fibrillary proteins was approximately fourfold for glycosaminoglycans and sixfold for proteoglycans, when compared with the conventional unhairing process. The enzyme application was successful for hide treatment, suggesting that this enzymatic preparation can be used in an environment-friendly leather production to replace the conventional chemical process.