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Over the course of 78 days, nine outdoor mesocosms, each with 1350 L capacity, were situated on a pontoon platform in the middle of a lake and exposed to 0 μg L−1 TiO2, 25 μg L−1 TiO2 or 250 μg L−1 TiO2 nanoparticles in the form of E171 TiO2 human food additive five times a week. Mesocosms were inoculated with sediment, phytoplankton, zooplankton, macroinvertebrates, macrophytes and fish before exposure, ensuring a complete food web. Physicochemical parameters of the water, nutrient concentrations, and biomass of the taxa were monitored. Concentrations of 25 μg L−1 TiO2 and 250 μg L−1 TiO2 caused a reduction in available soluble reactive phosphorus in the mesocosms by 15 and 23%, respectively, but not in the amount of total phosphorus. The biomass of Rotifera was significantly reduced by 32 and 57% in the TiO2 25 μg L−1 and TiO2 250 μg L−1 treatments, respectively, when compared to the control; however, the biomass of the other monitored groups—Cladocera, Copepoda, phytoplankton, macrophytes, chironomids and fish—remained unaffected. In conclusion, environmentally relevant concentrations of TiO2 nanoparticles may negatively affect certain parameters and taxa of the freshwater lentic aquatic ecosystem. However, these negative effects are not significant enough to affect the overall function of the ecosystem, as there were no cascade effects leading to a major change in its trophic state or primary production.