The increasing occurrence of disease, mortality, and antibiotic resistance in both animals and plants has prompted researchers to turn their focus toward environmentally sustainable methods for manufacturing metal oxide nanoparticles. These methods are valued for their simplicity and eco-friendliness compared to traditional chemical and physical approaches. This study presents a novel technique for synthesizing copper oxide nanoparticles (CuO NPs) utilizing an eco-friendly reducing agent, specifically Raphanus sativus leaf extract. Grounded in biological principles, this method offers a fresh perspective on nanoparticle synthesis. To verify the shape, size, and various bands present in the sample, different instruments, like UV–Vis spectroscopy for optical properties, Fourier-transform infrared spectroscopy (FTIR) for finding vibration energy of different bonds, and analysis size by using a zeta potential and particle size analyzer were used. Furthermore, the antimicrobial activity of CuO NPs was tested again with gram-positive bacteria such as Staphylococcus aureus, Streptococcus pyogenes, and gram-negative bacteria Escherichia coli. As a result, the synthesized CuO NPs nanoparticles exhibit promise as antimicrobial agents, suggesting their potential utility in the medical field in the future.