The green methodologies of nanoparticles with plant extracts have received an increase of interest. Copper oxide nanoparticles (CuO NPs) have been utilized in a many of applications in the last few decades. The current study presents the synthesis of CuO NPs with aqueous extract of Morinda citrifolia as a stabilizing agent. The leaf extract of Morinda citrifolia was mixed with a solution of copper sulphate (CuSO₄·5H₂O) and sodium hydroxide as a catalyst. UV–visible spectroscopy, FTIR, XRD, SEM, TEM, and EDAX analysis were performed to study the synthesized CuO NPs. Particle size distribution of the synthesized CuO NPs have been measured with dynamic light scattering. The CuO NPs synthesized were highly stable, sphere-like, and have size of particles from 20 to 50 nm. Furthermore, as-formed CuO NPs shown strong antibacterial activity against the Gram-positive bacteria (Bacillus subtilis, and Staphylococcus aureus), and Gram-negative bacteria (Escherichia coli). CuO NPs revealed a similar trend was analysed for antifungal activity. The zone of inhibition for the fungi evaluated for Aspergillus flavus (13.0 ± 1.1), Aspergillus niger (14.3 ± 0.7), and Penicillium frequentans (16.8 ± 1.4). According to the results of this investigation, green synthesized CuO NPs with Morinda citrifolia leaf extract may be used in biomedicine as a replacement agent for biological applications.