Breast cancer is the most common malignancy among women worldwide, and researchers are working to discover effective treatments to eradicate breast cancer. In the present study, we prepared PVP-functionalized palladium nanoparticles (PVP-PdNPs) for the treatment of human breast cancer MCF7 cells. Initially, the PVP-functionalized PdNPs were synthesized by an in situ method and confirmed with DRS-UV spectrometric analysis. Further, FTIR and Raman spectroscopic analyses showed the association of PVP with PdNPs by showing the vibrational mode of the PdNPs and C[double bond, length as m-dash]O stretch and CH2 band modes of PVP. Microscopic analysis showed that the PVP-PdNPs have a narrow size distribution with spherical shapes and a size range between 9 and 15 nm. The SAED and XRD patterns confirmed that the crystalline structure is face-centered cubic in nature, and EDAX mapping confirmed the formulation of PVP on the surface of the PdNPs. Further, in vitro MTT assay analysis showed that the PVP-PdNPs exhibit excellent cytotoxic activity against human breast cancer MCF7 cells in a dose-dependent manner. The PVP-PdNPs generate continuous ROS in the mitochondria; this leads to the damage of the mitochondrial membrane potential and nuclear DNA and induces apoptosis through caspase3/7 enzymatic activity. Together, the PVP-PdNPs are a promising potential anticancer agent against human breast cancer.