Concerning the increasing demand for antibacterial nature-based textiles, this work deals with the modification of cotton fabric with corn silk as a natural polymer together with the in situ synthesis of copper (II) oxide nanoparticles.Copper acetate is used as a precursor of copper (II) oxide and ascorbic acid as an environmentally friendly reducing agent. The copper (II) oxide/corn silk nanocomposite cotton fabrics are evaluated by FESEM, XRD, FTIR, water absorption, and antibacterial analysis. The treated fabrics exhibit higher water absorption and FESEM images along with EDX spectra confirm the formation of copper (II) oxide nanoparticles and corn silk layer on the fabric surface. FTIR analysis also establishes effective assembling of CuO/corn silk on the cotton surface. Moreover, the antimicrobial efficiency of the nanocomposite is verified to be higher than 90% against Gram-negative and Gram-positive model bacteria comprising Escherichia coli and Staphylococcus aureus, and antifungal properties against Candida albican. Consequently, the fabrication of copper (II) oxide/corn silk nanocomposites on the cotton fabric could be potentially applied for the preparation of antibacterial medical textiles.