This study investigated the galangin-loaded chitosan nanoparticles (GLN-CHNPs), which impede the proliferation of MCF-7 breast cancer cells. The successful incorporation of galangin into chitosan nanoparticles was confirmed using atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The synthesized GLN-CHNPs were then exposed to MCF-7 cells for 24 h to assess their cytotoxicity using the MTT assay; mitochondrial damage was analyzed using MitoSOX staining, and cell cycle stages were scanned by flow cytometry. Also, the protein expression of cell cycle regulatory markers and cyclin-related markers was observed via western blot analysis. The results exhibited that the GLN-CHNPs' average particle size was 164.2 nm and its zeta potential was 0.3 mV, indicating colloidal stability and their suitability for breast cancer treatment. XRD and SEM analyses confirmed the crystalline structure of GLN-CHNPs, while FTIR studies identified multiple stretching biomolecules, indicating the presence of various active compounds. Treatment with GLN-CHNPs led to significant cytotoxicity and mitochondrial damage in MCF-7 cells when compared with untreated cells (p ≤ 0.05). Additionally, GLN-CHNPs prompted cell cycle arrest that was observed at the G0-G1 phase accumulations (p ≤ 0.05) and inhibited the expression of cell cycle-regulating and proliferative markers. These findings advocate that GLN-CHNPs are an auspicious candidate for treatment of breast cancer as they effectively inhibit cell cycle progression and reduce proliferation. Further in vitro investigations using other BC cell lines are necessary to validate these findings.