Technical Abstract: The antimicrobial activity of lysozyme derives from the hydrolysis of the bacterial cell wall polysaccharide at the glycosidic bond that links N-acetyl-glucosamine and N-acetyl-muramic acid. Maintaining the activity of lysozyme while bound to a cellulose substrate is a goal toward developing enzyme-based antimicrobial materials. Here we outline the covalent attachment of lysozyme to three different types of glycine derivatized cotton materials as a model for its potential antimicrobial products. Lysozyme was immobilized on cotton printcloth, twill, and spunlaced nonwoven using three separate synthetic routes to enzyme immobilization. The attachment of lysozyme to cotton fibers was made through a glycine amino acid linker esterified to the cotton cellulose. The conjugation of lysozyme to cotton twill using a carbodiimide-mediated coupling reaction gave the highest incorporation of lysozyme and highest activity (8.9 micromoles/milligram cotton). A cotton spunlaced nonwoven gave slightly lower incorporation of lysozyme activity (7 micromoles/milligram cotton), and woven printcloth yielded less activity (2.2 micromoles/milligram cotton) utilizing the carobdiimide-mediated coupling approach. A carbonyldiimidazole-mediated coupling approach of lysozyme demonstrated significantly less incorporation compared with the carbodiimide-coupling, and comparable levels of lysozyme incorporation were found among the three fabrics with carbonydiimidazole coupling. The paper will discuss the resulting activities of the cotton fabrics and the relative advantages of the two immobilization chemistries used to link the lysozyme.