Barium titanate (BaTiO3) nanoparticles (BTNPs) have been considered as emerging materials in biomedical sector through last decades due to the excellent physicochemical properties such as dielectric and piezoelectric structures, biocompatibility, and nonlinear optical characteristics. In this study, BTNPs were synthesized via the co-precipitation method using barium carbonate and titanium dioxide by stirring for 5 h. Then, it was annealed at 850 °C for 5 h with five different concentrations: 0.2, 0.4, 0.6, 0.8, and 1 g/mL. The structural, morphological, and optical analyses were demonstrated by different characterization techniques such as X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray (EDX), thermogravimetric analysis (TGA), Raman, and UV–visible spectroscopy. The perovskite phase of BTNPs, an intense peak at 31.6°, was observed at the lowest concentration (0.2 g/mL), and the average crystalline size was 1.42 nm based on XRD pattern. The results have been justified by SEM and EDX. TGA demonstrated the adequate thermal stability of this material. EDX analysis confirmed the composition of Ti, Ba, and O elements. Raman peaks at 305 cm−1 and 517 cm−1 confirmed the formation of BaTiO3. UV–visible spectra presented that its’ absorbance edge shifted into visible range at 404 nm. Application of BTNPs on breast cancer cell line (MCF-7) presented significant dispersion effect at 0.2, 0.4 and 0.6 g/mL of BaTiO3. A strong toxicity rate of BaTiO3 has been observed against the MCF-7 cell line. Maximum % of cell viability loss, $$\cong$$ ≅ 57% was recorded at 200 µg/mL of BTNPs, and minimum % of cell viability loss was observed as 19% at 50 µg/mL of BTNPs. The results presented that a higher concentration of BTPNs dosage was more effective in inhibition of breast cancer cells. Therefore, BTNPs can be recommended as a promising nanomaterial for anti-cancer drug discovery.