The rapid development of nanotechnology has significantly transformed various industrial processes associated with food production (e.g., through the introduction of smart and active packaging, nanosensors, nanopesticides, and nanofertilizers). This study focuses on two nanoparticles widely used in the food industry, titanium dioxide (TiO₂-NPs) and zinc oxide (ZnO-NPs), and their effects on human cells. Thus, the cytotoxicity of TiO₂-NPs (commercially acquired; spherical shape with an average size of 298.4 nm and a PDI of 0.248) and ZnO-NPs (synthesized in the laboratory; spherical-like shape with an average size of 339.9 nm and a PDI of 0.590) to a human colon cancer cell line (HCT116) was studied, assessing cellular metabolic activity, as an indicator of cell viability, through the MTT assay. Overall, exposure to ZnO-NPs induced a dose-dependent reduction in cell viability, with half maximal inhibitory concentration (IC₅₀) values of 10.4 mg/L, 8.8 mg/L, and 7.7 mg/L at 24, 48, and 72h, respectively. In contrast, TiO₂-NPs did not induce significant differences in cell viability across the same time points. These findings highlight the differential cytotoxic effects of ZnO-NPs and TiO₂-NPs on colon cancer cells, suggesting a need for the careful consideration of ZnO-NPs in food applications, due to their potential health risks. Overall, this study provides crucial insights into the biological interactions of these nanoparticles, highlighting the importance of thorough safety evaluations in their use within the food industry.