Concerns about the possible ecotoxicological implications of nano-sized plastic materials in the freshwater environment are growing with the increasing use of plastic materials. The present study focuses on the behavior and effects of amidine-functionalized polystyrene (NPLs) of 20, 40, 60, and 100-nm-size in freshwaters and different synthetic media. Daphnia magna was exposed to increasing concentrations from 0.5 to 30 mg/L (and from 0.5 to 100 mg/L for 100-nm-sized NPLs). The results revealed no significant aggregation in ultra-pure water, culture media, and synthetic water. In the presence of natural organic matter, NPLs of 20 and 40 nm displayed better stability in both freshwater and synthetic media, whereas a significant aggregation of 60 and 100 nm PS NPLs was found. All the studied PS NPLs with size between 20 and 100 nm exhibited acute toxicity to D. magna. The observed 48-h immobilization strongly depended on the primary size of PS NPLs, with 20 and 40-nm-size PS NPLs inducing a stronger effect in both freshwaters and synthetic media. Water quality variables such as pH, cation and anion composition, and DOC were of secondary importance. The results of the present study confirmed the toxicity of NPLs of different sizes to crustaceans in natural freshwater and synthetic media and demonstrated the importance of the primary size of NPLs in the behavior and effects of NPLs.