The aim of this work was to investigate the combined short-term toxic effect of zinc oxide (ZnO) nanoparticles (NPs) and nitrate concentration of the medium on freshwater microalgae. For this purpose, freshwater microalgae Chlorococcum sp. was cultivated in modified Blue-Green medium (BG-11) containing nitrate concentrations ranging from 0 to 300 mg/L, and exposed to ZnO NPs in different concentrations (0.081 to 810 mg/L) for a period up to 96 h. The experimental results revealed that algal growth was affected by the exposure time, NPs concentrations, and mainly the initial nitrate concentration. Differences in microalgae growth rates were observed. The toxic effect of ZnO NPs was higher on microalgae cultured in modified BG-11 with low and high nitrate concentrations. During the 4-day exposure, the highest growth rates were observed at 24 h at an initial nitrate concentration of 50 mg/L; 1.94 d−1 and 0.22 d−1 for 0 and 810 mg/L ZnO NPs, respectively. Nitrate uptake by algal biomass reached up to 40.1% after 96 h of operation in the control culture with an initial nitrate concentration of 50 mg/L. Finally, the results of this study showed the need for the investigation of ZnO NPs toxicity on microalgae under optimum and stressful nutrient conditions for microalgae growth.