| Summary: | The leaf extract of <i>Indigofera linnaei</i> Ali, an Indian medicinal plant, was utilized in the synthesis of copper oxide nanoparticles (CuO-NPs). Green chemistry is a safe and cost-effective method for the synthesis of nanoparticles using plant extracts. The synthesis of CuO NPs was confirmed using ultraviolet–visible (UV-visible) spectrum λ-max data with two peaks at 269 and 337 nm. Different functional groups were identified using Fourier-transform infrared spectroscopy (FT-IR). X-ray diffraction (XRD) was used to confirm the crystalline structure of the CuO-nanoparticles. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analyses were performed to examine the surface morphology and elemental composition of the biosynthesized CuO-NPs. Furthermore, the synthesized CuO-NPs exhibited antibacterial activity against <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, <i>Escherichia coli</i>, and <i>Enterococcus faecalis</i>. Additionally, they exhibited a good insecticidal effect on <i>Culex quinquefasciatus</i> larvae, with low LC<sub>50</sub> 55.716 µg/mL and LC<sub>90</sub> 123.657 µg/mL values. The CuO-NPs inhibited human breast cancer cells in a concentration-dependent manner, with an IC<sub>50</sub> value of 63.13 µg/mL.
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