This study reports the synthesis of Cobalt ferrite (CoFe2O4) nanoparticles (NPs) by a green synthesis method using the leaf extract of Barleria Lupulina. The X-ray diffraction (XRD) analysis confirms the formation of a single-phase cubic (Fd3m) structure and from the Scherrer formula the average crystallite size (30.07 nm) calculated matches with that obtained from W-H plot(34.66 nm). A bandgap of 2.02 eV was observed from the UV–Vis-DRS analysis. Fourier transform infrared spectroscopy (FTIR) was used to confirm the characteristic functional groups present in the spinel ferrite. The investigation using scanning electron microscopy (SEM) shows aggregates of nanoparticles and EDX analysis gives the chemical composition of the green synthesized ferrite sample. The distribution of particle-sizes and colloidal-stability were estimated by DLS and zeta-potential measurements. The M−H plot confirmed the ferromagnetic nature of the prepared nanoparticles. Beyond the realm of material science, we ventured into the realm of biological activity. Our synthesized cobalt ferrite nanoparticles were assessed for their toxicity on human breast cancer cell lines (MCF-7) as well as normal L929 fibroblast cell lines by MTT assay. The results show potent anti-cancer activity of the ferrite sample (LC50 value 77.92 µg/ml) while sparing normal cells from excessive toxicity (LC50 value 252.34 µg/ml). The results of cell viability, changes in cell morphology, and cell cycle analysis revealed that cobalt ferrite nanoparticles triggered apoptosis, and hindered cell adhesion and migration of MCF-7 cells, potentially through elevated reactive oxygen species generation. This dual action, where the nanoparticles combat cancer effectively while minimizing harm to healthy cells, holds significant promise in the field of oncology.