The probable underlying mechanism(s) of bacterial cell-TiOnanoparticles (TiONPs) interaction in the absence of photo-irradiation has been less studied since most of the prior cytotoxicity studies focused on irradiated TiO The present study draws attention to the possible role of cell surface-TiONP interactions under dark conditions, through an array of spectroscopic and microscopic investigations. A dominant freshwater bacterial isolate, Bacillus licheniformis, which interacted with environmentally relevant concentrations of TiONPs (1 μg/mL), was analysed and compared under both light and dark conditions. Aggregation of cells upon NP interaction and adsorption of NPs onto the cell membrane was evident from the scanning electron micrographs under both light and dark conditions. The FT-IR and FT-Raman spectra suggested stress response of bacterial cells by elevated protein and polysaccharide content in the cell-NP interaction. The Xray photoelectron spectroscopic data substantiated the reduction of titanium from Ti(IV) to Ti(III) species which might have contributed to the redox interactions on the cell surface under light as well as dark conditions. The internalization of NPs in the cytoplasm were obvious from the transmission electron micrographs. The consequent cell death/damage was confirmed through fluorescence spectroscopy and microscopy. To conclude, the current study established the substantial role of interfacial interactions in cytotoxicity of the TiONPs irrespective of the irradiation conditions. © Indian Academy of Sciences.