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The study synthesized Pluronic F-127 nanoparticles that encapsulate Fe2O3 (PF127Fe2O3NPs), nanoparticles, characterized their formation, and evaluated their cytotoxicity and anticancer activity using Berberis vulgaris leaf extract, using various analytical methods such as FTIR, Ultraviolet-visible, photoluminescence, dynamic light scattering, X-ray diffraction, and morphology analysis. We assessed the antioxidant properties of PF127Fe2O3NPs, cytotoxicity, and apoptosis through 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and acridine orange/ethidium bromide staining in breast cancer cells, such as MCF7, and MDA-MB-231. The characterization results demonstrated that PF-127 was coated with Fe2O3 nanoparticles. MTT assay data revealed that PF127Fe2O3NPs effectively prevent cancer cells from proliferating and act as an anticancer drug. The antimicrobial results revealed that the fabricated nanoparticles are effective against gram-negative (Klebsiella pneumoniae, Escherichia coli, and Shigella dysenteriae) and gram-positive (Streptococcus pneumoniae, Staphylococcus aureus, and Bacillus subtilis) bacteria. Treatment of PF127Fe2O3NPs in a dose-dependent manner on MCF7, and MDA-MB-231, exhibited increased antioxidant activity, nuclear damage, and apoptotic activity. These results confirm the apoptotic activity of PF127Fe2O3NPs. The study concludes that MCF7 appears to be more sensitive to PF127Fe2O3NPs than MDA-MB-231. In conclusion, we have found that it can be used as an effective antioxidant and anticancer agent in therapeutics.