Textiles functionalized with copper oxide nanoparticles (CuO NPs) have become a favourable material to inhibit the spread of diseases due to their anti-microbial properties. In the present work, a successful procedure for in situ growth of CuO NPs in textiles was developed. Results showed that the combination of in situ synthesis and pad-dry-cure method promoting a uniform and dense adsorption of nanoparticles on the surface of the fabrics. The CuO NP-coated fabrics was characterized using XRD, SEM, EDX, TEM, and FTIR techniques. The CuO NP-coated cotton fabrics showed better anti-bacterial activity against various bacteria's namely Staphylococcus aureus, Escherichia coli, Pseudomonas fluorescens and Bacillus subtilis. Besides, it also showed better antifungal activity against Candida albicans. The CuO NPs impregnated cotton fabrics exhibit a bacterial reduction of more than 90%, which is sustainable even after 20 washing cycles. Therefore, the CuO NP-coated fabrics has great potential to be used as coatings for medical, cosmetic or sports fabrics.