Titanium dioxide nanoparticles (TiO2NPs) have been widely used in food, cosmetics, and biomedical research. However, human safety following exposure to TiO2NPs remains to be fully understood. The aim of this study was to evaluate the in vitro safety and toxicity of TiO2NPs synthesized via the Stöber method under different washing and temperature conditions. TiO2NPs were characterized by their size, shape, surface charge, surface area, crystalline pattern, and band gap. Biological studies were conducted on phagocytic (RAW 264.7) and non-phagocytic (HEK-239) cells. Results showed that washing amorphous as-prepared TiO2NPs (T1) with ethanol while applying heat at 550 °C (T2) resulted in a reduction in the surface area and charge compared to washing with water (T3) or a higher temperature (800 °C) (T4) and influenced the formation of crystalline structures with the anatase phase in T2 and T3 and rutile/anatase mixture in T4. Biological and toxicological responses varied among TiO2NPs. T1 was associated with significant cellular internalization and toxicity in both cell types compared to other TiO2NPs. Furthermore, the formation of the crystalline structure induced toxicity independent of other physicochemical properties. Compared with anatase, the rutile phase (T4) reduced cellular internalization and toxicity. However, comparable levels of reactive oxygen species were generated following exposure to the different types of TiO2,indicating that toxicity is partially driven via non-oxidative pathways. TiO2NPs were able to trigger an inflammatory response, with varying trends among the two tested cell types. Together, the findings emphasize the importance of standardizing engineered nanomaterial synthesis conditions and evaluating the associated biological and toxicological consequences arising from changes in synthesis conditions.