Raw cotton fabrics contain natural impurities (fats, waxes, pectins, and proteins), which impart hydrophobic nature and interfere with textile dyeing. Typically, these noncellulosic impurities are removed by alkaline scouring with hot aqueous sodium hydroxide. As a greener alternative with reduced environmental impact, enzymes can be used to improve cotton hydrophilicity and the quality of fabrics. Thus, this study aimed to establish a roadmap for bioscouring of knitted cotton fabrics using a commercial pectinase preparation under mild conditions of pH and temperature. Pectate lyase (1–10 g L−1) was applied for 30 min at 55 °C at optimum pH 8.5, evaluating different post-treatments with varying temperature and surfactant concentration. The treated samples were characterized in terms of weight loss, water absorption, pectin content, whiteness index, and FTIR spectra. A high level of cleaning was achieved, with an efficacy comparable to conventional alkaline scouring. It was possible to increase the wettability of the textile article with a reduction in fiber damage and in the consumption of energy, water, and chemicals. Low surfactant concentration (0.2 g L−1) and 70 °C washing temperature in the post-washing step were found sufficient to aid in the removal of waxes and fats from the cotton surface after bioscouring and were adequate to achieve high hydrophilicity at low weight loss, unlike other bioscouring studies that used temperatures equal or higher than 80 °C. Results clearly proved that the efficiency of bioscouring to achieve suitable wettability of knitted cotton fabrics depends essentially on an effective post-washing step and the selected conditions, which impact the sustainability and economic viability of the whole process.