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The present study evaluated the anticancer potential of copper oxide nanoparticles (CuO NPs) synthesized from pumpkin seed extract in human breast cancer cell line (MDA‐MB‐231) using a battery of tests such as MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay, morphological alteration, reactive oxygen species (ROS) generation, and changes in mitochondrial membrane potential (MMP). The biogenic CuO NPs showed a dose‐dependent decline in cell viability with 50% inhibitory concentration (IC50) at 20 μg/ml. Treatment with an IC50 dose of CuO NPs resulted in considerable morphology changes, such as shrinkage, detachment, membrane blebbing, and deformed shape in MDA‐MB‐231 cells. We also observed a significant dose‐dependent increase in ROS production and MMP modulation due to CuO NP treatment. Overall, CuO NPs showed significant anticancer potential in …