Pectinases can be used to remove impurities and hydrophobic material from cotton fibers by the degradation of pectic substances. The biochemical characterization and the evaluation of process parameters that influence the enzyme's activity are mandatory to optimize the industrial application. In the present study, a factorial experimental design was conducted to evaluate the activity of a commercial pectate lyase at different reaction conditions of temperature, pH, and nonionic surfactant concentration, aiming its application in bioscouring of textiles. The main effects of all three factors were found to be statistically significant. The second-order interactions between temperature and pH, and pH and surfactant concentration, were also important. Maximum activity on polygalacturonic acid was achieved at 60 °C, pH 9, and without surfactant. These results provided support for the subsequent bioscouring assays, resulting in a high level of cleanliness and good wettability of knitted cotton fabric at the lowest enzyme concentration (1 g/l), and could make textile processing more sustainable.