To better understand the potential impacts of metal oxide nanoparticles (NPs) on Gram(+) Bacillus subtilis and Gram(-) Escherichia coli (K12) bacteria, eight different nanosized titanium dioxide (TiO2) suspensions with five different concentrations were used. Water quality parameters (pH, temperature, and ionic strength), light sources, and light intensities were also changed to achieve different environmental conditions. The photosensitive TiO2 NPs were found to be harmful to varying degrees under ambient conditions, with antibacterial activity increasing with primary particle sizes from 16 to 20 nm. The presence of light was a significant factor under most conditions tested, presumably due to its role in promoting generation of reactive oxygen species (ROS). However, bacterial growth inhibition was also observed under dark conditions and different water quality parameters, indicating that undetermined mechanisms additional to photocatalytic ROS production were responsible for toxicity. The results also indicated that nano-TiO2 particles in the absence and the presence of photoactivation induced lipid peroxidation and cellular respiration disruption.