ZnO nanoparticles (NPs) synthesis on cotton fabric through the formation of biologically activated ammo-nia from urea broth in the presence of the ureolytic bacterial species Serratia ureilytica (HM475278) hasbeen described in the present contribution. The cotton fabric was immersed in biogenic zinc ammoniumcomplex medium and subjected to heat treatment at an optimum temperature of 50◦C for different dura-tions of time (30, 60, 90 min). The crystal growth of ZnO nanoparticles on cotton fabric was characterizedby analytical techniques such as SEM-EDAX, XRD, TGA, CHNS and UV–visible spectra, and evaluation ofantibacterial activity was carried out against Escherichia coli and Staphylococcus aureus. Crystal growth andmorphological studies confirmed the attachment of ZnO NPs on the cotton fabric. Spherical to nanoflowershaped particles were obtained with increasing time duration from 30 to 90 min. The antibacterial activ-ity of loaded cotton fabrics was found to be substantially higher than the bare cotton samples. Wet filminterfacial contact studies have shown greater antibacterial activity as a result of nanoparticle contact atthe bio-interface, as observed by Epi-fluorescent microscopic observations.