The use of immobilized enzymes in enhancing the biodegradability of cotton textiles is of great interest for sustainable textile waste management. Understanding how enzyme activity influences biodegradation rates is crucial for optimizing textile disposal strategies. This study aims to assess the impact of enzyme immobilization on the biodegradability of cotton fabrics, compared to microcrystalline cellulose and cotton treated with both active and inactivated enzymes. Biodegradability was evaluated by measuring CO₂ production over 75 days when the fabrics were buried in soil, as a proxy for microbial degradation. The study compared untreated cotton, cotton treated with immobilized enzymes, cotton with inactivated enzymes (via thermal treatment), and microcrystalline cellulose as reference. CO₂ emissions were monitored to quantify the biodegradation levels in each sample. The results indicated that microcrystalline cellulose exhibited the lowest biodegradability, with significantly lower CO₂ production. Among the cotton samples, the highest biodegradability was observed in the fabric treated with immobilized enzymes that had been inactivated by heat. This was followed by the cotton treated with active immobilized enzymes, while untreated cotton exhibited the lowest biodegradability of all the cotton samples. Although enzyme immobilization can enhance the stability and sustainability of degradation processes, thermal inactivation of enzymes unexpectedly increased biodegradation rates in cotton fabrics. This suggests that not only enzymes but others, such as structural changes in the cotton fibers, may play a role in facilitating biodegradation. Further research is needed increasing time and to clarify the mechanisms involved and optimize immobilization techniques for textile waste management.