The scope of this study was to investigate the toxic effects of zinc oxide (ZnO) nanoparticles (NPs) on freshwater microalgae, in long-term semi-batch feeding mode at two different hydraulic retention times (HRTs) (20 and 40 days). A freshwater microalgae, Scenedesmus rubescens, was employed and exposed to a semi-continuous supply of ZnO NPs at a low concentration of 0.081 mg/L for a period of 28 d. Experiments were conducted under controlled environmental conditions. Τhe impact of ZnO NPs on S. rubescens, which was assessed in terms of nutrient removal, biomass growth, and algal lipid content. Semi-batch mode cultures showed that low ZnO NP concentrations at an HRT of 40 d did not have any negative effect on microalgae growth after the fourth day of culture. In contrast, algal growth was inhibited up to 17.5% at an HRT of 20 d in the presence of ZnO NPs. This might be attributed to the higher flow rate applied and ZnO NPs load. A positive correlation between nutrient removal and microalgae growth was observed. The algal lipid content was, in most cases, higher in the presence of ZnO NPs at both HRTs, indicating that even low ZnO NPs concentration cause stress resulting in higher lipid content.