Titanium dioxide (TiO2) nanopowders at different concentrations (0–50 mg L−1) were injected into an aerobic-sequencing batch reactor (SBR) to investigate the effects of long-term exposure to nanoparticles on bacterial and protozoan communities. The detection of nanoparticles in the bioflocs was analyzed by scanning electron microscopy, transmission electron microscopy, and energy-dispersive x-ray spectroscopy. The SBR wastewater experiments were conducted under the influence of ultraviolet light with photocatalytic TiO2. The intrusion of TiO2 nanoparticles was found both on the surface and inside of the bioflocs. The change of microbial population in terms of mixed liquor-suspended solids and the sludge volume index was monitored. The TiO2 nanoparticles tentatively exerted an adverse effect on the microbial population, causing the reduction of microorganisms (both bacteria and protozoa) in the SBR. The respiration inhibition rate of the bacteria was increased, and the viability of the microbial population was reduced at the high concentration (50 mg L−1) of TiO2. The decreasing number of protozoa in the presence of TiO2 nanoparticles during 20 days of treatment with 0.5 and 1.0 mg L−1 TiO2 is clearly demonstrated. The measured chemical oxygen demand (COD) in the effluent tends to increase with a long-term operation. The increase of COD in the system suggests a decrease in the efficiency of the wastewater treatment plant. However, the SBR can effectively remove the TiO2 nanoparticles (up to 50 mg L−1) from the effluent.