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Due to the potential of nanotechnology in suppressing the increasing burden of pathogenic infections and cancer incidences, researchers are developing nanoparticles that are high in efficacy and economically affordable. Herein, we aimed to synthesize Cu nanoparticles (CuNPs) via a green route by utilizing Annona squamosa L stem barks and evaluate its activities against pathogenic bacteria and fungi (Staphylococcus aureus, Escherichia coli, and Candida albicans). The aqueous extract of Annona squamosa L stem barks was determined for total phenolic content (TPC) and total flavonoid content (TFC). The CuNPs were obtained by adding the CuSO4 0.1 M into the foregoing extract and further characterized using Fourier-transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy, transmission electron microscopy, and X-Ray diffraction. The antibacterial and antifungal activities were examined based on Kirby-Bauer method. The anticancer potential of CuNPs were assessed based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using breast cancer MCF-7 cells. The results revealed that the spherical CuNPs had average particle diameter of 13.7 ± 3.3 nm and was observed to interact with the phytoconstituents contained in the A. squamosa extract. Inhibition zones of 15.7, 12.3, and 13.9 mm were achieved by the CuNPs against S. aureus, E. coli, and C. albicans, respectively. As for the anticancer activity (MCF-7), the IC50 of 130.39 mg/L was obtained. In conclusion, the A. squamosa stem barks-mediated CuNPs could act as an anti-pathogenic agent with anticancer activity requiring further investigation using different cancer cells.