Titanium dioxide nanoparticles (TiO NPs) are widely distributed in aquatic environments due to their extensive industrial and commercial applications. Several studies have reported the adverse effects of TiO NPs on aquatic organisms; however, limited information is available regarding their impact on the freshwater mussel Lamellidens marginalis. The present study investigates the physiological and biochemical responses of L. marginalis following acute exposure (7 days) to varying concentrations of TiO NPs (Control, 5.0, 50, and 100 µg/L). Physiological parameters, including the condition index, filtration rate, and clearance rate, were assessed alongside hemocyte count, metabolic activity (electron transport system activity), and energy reserve content (glycogen, lipid, and protein levels). Additionally, oxidative stress biomarkers, including antioxidant enzyme activity, biotransformation enzyme activity, and lipid peroxidation levels, were evaluated. Results revealed a significant accumulation of TiO NPs in the gill tissues, accompanied by a marked decline in filtration rate and total hemocyte count, along with an increase in nitric oxide production. Exposure to higher concentrations of TiO NPs resulted in substantial alterations in energy reserve levels and oxidative stress biomarkers, indicative of metabolic disruption. Furthermore, mussels exposed to elevated TiO NP concentrations exhibited reduced feeding activity and energy expenditure, leading to impaired physiological performance, including potential consequences for growth and reproduction. Histopathological analysis demonstrated pronounced gill damage in mussels from the higher exposure groups. These findings emphasize the ecological risks associated with TiO NP contamination and underscore the need for stringent measures to mitigate their impact on freshwater bivalves.