Technical Abstract: Covalent attachment of enzymes to cellulosic materials like cotton is of interest where either release or loss of enzyme activity over time needs to be avoided. The covalent attachment of an enzyme to a cellulosic substrate requires either activation of a protein side chain or an organic functional group on the cellulosic substrate. Use of a water soluble carbodiimide to create an amide linkage as the covalent attachment between the enzyme and substrate represents an aqueous-based alternative which may be preferred for textiles processes. Here we describe an amide bond-mediated lysozyme immobilization applied to cotton where either the carboxylate side chains of the protein or pendant carboxylates in a citrate, cross-linked cotton support are activated as the O-acyl-isourea intermediate, and the reactive amino nucleophiles are derived from amino-silanized cotton and the protein’s amino side chains, respectively. A comparison is made of the two actvation approaches to covalently link lysozyme to two different cotton fabrics using the water soluble carbodiimide 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate. A comparison of the resulting enzyme activities of lysozyme on two different cotton supports showed that linking lysozyme to citrate crosslinked cotton gave higher activity than on aminosilanized cotton. The lysozyme-celluose conjugate formed on the citrate crosslinked nonwoven cotton fabric gave the highest yield and antimicrobial activity.