In this work, TiO2 nanostructures (TiO2-NSs) were obtained at low temperature, using the sol–gel method, and their toxicity was evaluated in Swiss albino mice. For the synthesis, ethanol solvent was used and the concentration of the surfactant, cetyltrimethyl ammonium bromide (CTAB), was modified to determine its effect on the characteristics of the final product. Characterization of the synthesized TiO2-NSs was carried out using IR spectroscopy, X-ray diffraction (XRD), transmission (TEM) and scanning electron microscopy (SEM). The solids obtained, without heat treatment, showed an amorphous structure. When these were treated at 450 °C, they crystallized in an anatase type phase, independent of CTAB concentration, with a crystallite size of 3–5 nm. The particles showed a spheroidal or needle-like shape, with a nanometric size (100 nm), and formed spheroidal agglomerates (between 1 and 5 μm) or mesoporous structures, for the 6 mm concentration of CTAB. Additionally, the toxicity of the NSs synthesized was determined, using 3 mm CTAB and treated at 450 °C, giving doses of 5 mg/kg, 50 mg/kg, 300 mg/kg of TiO2-NSs to mice prepared for this test. The results of the analysis of the liver biopsies of the animals, on which euthanasia was practiced, showed that the best effect was the 300 mg/kg dose, where alterations in the mitochondria were observed, with distortion in their normal structure (fragmentation) and strong involvement of its outer membrane.