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Titanium dioxide nanoparticles (TiO2 NPs) are photo-active metallic nanoparticles becoming promising agents in modern biomedical applications. Herein, a novel process for the synthesis of TiO2 NPs with high stability was developed by a sol–gel process and to investigate their cytotoxicity and antibacterial activity. Numerous experiments have been performed to confirm the morphologies, compositions, and physicochemical properties of prepared TiO2 NPs, such as field emission scanning electron microscopy, dynamic light scattering, Zeta potential, Fourier transform infrared spectroscopy and X-ray diffraction. MTT assay was applied to assess the cytotoxicity of the prepared nanoparticles. The results indicate that the synthesized nanoparticles' diameter is about 68 nm and contains the anatase phase, in the range of 2θ from 25 to 80 °C. The hydrodynamic radius of nanoparticles is about 140.4 nm, and the zeta potential of nanoparticles is about −44.6 mV. The MTT results have not shown any toxicity; the antibacterial inhibitory effect of TiO2 NPs at 200 mg/mL concentrations exhibited superior antibacterial activity at 15.9 ± 0.1, 14.0 ± 0.1 against Staphylococcus aureus and Escherichia coli, respectively. In conclusion, colloidal solutions with high stability were successfully synthesized, contributing to decreased dimensions and increased antibacterial properties. [ABSTRACT FROM AUTHOR]