There has recently been an increase in the usage of TiO2 nanoparticles (NPs). P25 TiO2 NPs, a mixture of anatase and rutile phase in 3:1 ratio, are generally used for photocatalytic applications because both phases exhibit a synergistic effect on the photocatalytic activity of the TiO2 NPs. In the present study, increased toxicity of UVA-pre-irradiated P25 TiO2 NPs on freshwater algae Scenedesmus obliquus was assessed under visible light and dark exposure conditions at actual low concentrations (0.3, 3 and 35 μM of Ti). Photocatalytic property of P25 TiO2 NPs caused disaggregation of UVA-pre-irradiated NPs, thus significantly decreasing the mean hydrodynamic diameter (MHD) (188.74 ± 0.54 nm) than that of non-irradiated NPs (232.26 ± 0.44). This decrease in diameter of UVA-pre-irradiated NPs may increase its biological activity towards algal samples. All concentrations of pre-irradiated NPs, under both light and dark conditions, showed a significantly lesser cell viability (p