TiO2 nanoparticles (NPs), which are mainly used in consumer products (mostly cosmetics), have been found to cause ecotoxic effects in the aquatic environment. The green algae Raphidocelis subcapitata, as a representative of primary producers of the freshwater ecosystem, has been frequently used to study the effects of metal oxide NPs. An ecotoxicity study was conducted herein to investigate the effects of TiO2 NPs on survival and membrane deformation of algal cells. Five different concentrations of nano-TiO2 particles (1, 10, 50, 100 and 500 mg/L) were prepared in synthetic surface water samples with five different water quality characteristics (pH 6.4–8.4, hardness 10–320 mg CaCO3/L, ionic strength 0.2–8 mM, and alkalinity 10–245 mg CaCO3/L). Results showed a significant increase in the hydrodynamic diameter of NPs with respect to both NP concentrations and ionic content of the test system. A soft synthetic freshwater system at pH 7.3 ± 0.2 appeared to provide the most effective water type, with more than 95% algal mortality observed at 50, 100 and 500 mg/L NP concentrations. At high exposure concentrations, increased malondialdehyde formations were observed. Moreover, due to membrane deformation, TEM images correlated the uptake of the NPs.