The aim of this work was to investigate the long-term toxic effect of zinc oxide (ZnO) nanoparticles (NPs) on freshwater microalgae, combined with the nutrient consumption in the culture. For this purpose, two common microalgae media (Blue-Green 11, BG-11, and Bold's Basal Medium, BBM) were used. Scenedesmus rubescens was used as freshwater microalgae model species and was exposed to ZnO NPs at different concentrations (0.081 to 810 mg/L) for a period up to 28 d. The experimental results revealed that microalgae growth was affected by the time of exposure and the NPs concentrations, but mainly the culture medium used. Differences in microalgae growth rates were observed and attributed to the selected culture medium. The toxic effect of ZnO NPs was higher on microalgae cultured in modified BG-11 compared to BBM, despite the fact that S. rubescens exhibited higher growth rate in modified BG-11 without the exposure of ZnO NPs.