Copper oxide nanoparticles have attracted a great deal of attention in many fields due to their attractive physico‐chemical properties. In particular, their synthesis using an environmentally friendly method offers advantages in many applications. In the present report, copper oxide nanoparticles are produced from extracts of the plant Paulownia tomentosa by a method of biosynthesis. The synthesized CuO NPs have been characterized morphologically and structurally by fourier transform infrared spectroscopy, scanning electron microscopy, energy‐dispersive X‐ray analysis, and X‐ray diffraction. Furthermore, the antimicrobial, antibiofilm and anticancer activities of the synthesized CuO NPs were evaluated. The CuO NPs were spherical and the particle size was 12.5 nm. For the tested microorganisms, CuO NPs showed a high inhibitory effect in terms of antimicrobial and antibiofilm activity. The cytotoxicity of the synthesized CuO NPs against HT‐22 and MCF‐7 showed a dose‐response activity. 50 % inhibitory concentration values were found to be 18.34 and 14.32 μg/mL of CuO NPs against the HT‐22 and MCF‐7 cancer cells, respectively. The results suggest that these nanoparticles can be used as effective antibacterial, antibiofilm and anticancer agents in the biomedical fields.