Resistance to antibiotics is a global concern and urgently needs sincere attention. Looking toward the current scenario there is a definite need for hybrid systems which can combat with the pathogens. One such approach is synthesis of CuO NPs using cow urine for the first time. The advantage of this method is its eco-friendly and economical synthesis approach. It is seen that the prepared NPs exhibit excellent antimicrobial activity against test microorganism such as Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Also, the synthesized NPs show synergistic effectiveness with common broad spectrum antibiotic tetracycline. In addition, the prepared NPs are also tested for anticancer activity by using MTT assay and results suggests that the prepared NPs shows excellent anticancer activity against MCF7 cell lines. The developed material carries excellent potential in biomedical application. © 2020 Wiley-VCH GmbH