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The current work investigated the ecotoxicological effects induced by Titanium Dioxide (TiO₂) nanoparticles (NPs), used at three different concentrations (C1 = 10 μg·L⁻¹, C2 = 100 μg·L⁻¹ and C3 = 1000 μg·L⁻¹) in a laboratory experiment, on the freshwater mussel <i>Unio ravoisieri</i>. Biochemical analyses of gills and digestive glands revealed a stress-related disruption of the antioxidant system. The catalase activity and the rates of malonedialdehyde and hydrogen peroxide production were significantly higher in both organs following the exposure to TiO₂ NPs and was concentration-dependent. In addition, based on the observed changes in acetylcholinesterase activity, it can be concluded that the disturbance threshold for the cholinergic system was less than 1 mg·L⁻¹ of TiO₂. Overall, the results suggest that the mussel <i>Unio ravoisieri</i> could be used as a sentinel species in monitoring surveys assessing the environmental impact of metallic nanoparticles in freshwater systems.