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Textiles are known to be the best substrates for growing a variety of microorganisms efficiently at appropriate temperatures and humidity in contact with the human body. Currently, increasing public concern about hygiene has been driving many investigations about antimicrobial surface modification of textiles. The present research reports on the synthesis and characterization of nanosized copper oxide nanoparticles (CuONPs) and their application on cotton fabric to increase the bactericidal and hydrophobic properties. The synthesized materials have been subjected to spectroscopic and microscopic characterizations to help in understanding their structure, morphology, size, and composition. Further, upon dispersion of the nanoparticles onto the fabric, its hydrophobicity and mechanical properties were evaluated using electron microscopy and universal testing machine. Treated cotton fabric exhibits higher tensile strength (32 MPa) than the untreated one (27 MPa), whereas copper nanoparticle-coated cotton fabric shows a fair hydrophobicity. Moreover, CuONPs-treated and untreated cotton fabrics have been analyzed for bactericidal activity against various gram-negative and gram-positive strains. Finally, the CuONPs-coated cotton fabric displays greater antibacterial activity against E. coli and exhibits superior antimicrobial activity even after 30 cycles of washing, indicating that the CuONPs-coated cotton fabric has a higher potential to be employed as a medical textile to avoid cross-infection within a clinical environment.