In the current study cotton fabric was in situ loaded with zinc oxide nanoparticles. Urea was used as a dispersion medium to transform zinc acetate salt into ZnONPs within the matrix of the cotton fabric. These cotton fabrics were characterized before and after loading of ZnONPs using DTA, FTIR, SEM coupled with EDX, tensile strength and air permeability in order to determine the effect of the in situ loaded ZnONPs on the thermal behavior, chemical integrity, microstructure, mechanical properties and air permeability (%) properties of the cotton fabrics. The obtained results reviled that presence of in situ loaded ZnONPs has a slight effect on the thermal behavior of the cotton fabrics. Also, presence of in situ loaded ZnONPs was confirmed by FTIR and SEM coupled with EDX techniques in form of dispersed aggregates within the cotton matrix. The physical properties of the in situ loaded ZnONPs were found to be dependent on the concentration of the ZnONPs. This treated cotton fabrics are recommended for biomedical applications.