The ubiquitous nature of plastics, particularly nanoplastics, raises concern about their potential effects on primary producer microalgae. Currently, the impacts and potential mechanisms of nanoplastics on microalgae are not fully understood. In this study, the effects of two plain commercial polystyrene nanoplastics (PS-NPs) with different sizes (50 nm and 70 nm) onC. vulgariswere assessed in a concentration range of 0–50 mg/L during 72 h exposure periods. Results revealed that both PS-NPs have dose-dependent toxicity effects onC. vulgaris,as confirmed by the decrease of growth rates, chlorophyll a and esterase activities, and the increase of ROS, MDA, and membrane damage. The membrane damage was caused by the agglomeration of PS-NPs on microalgae and may be the key reason for the toxicity. Compared with 70 nm PS-NPs (72 h EC50>50 mg/L), 50 nm PS-NPs posed greater adverse effects on algae, with an EC50–72hof 19.89 mg/L. FTIR results also proved the stronger variation of macromolecules in the 50 nm PS-NPs treatment group. This phenomenon might be related to the properties of PS-NPs in exposure medium. The lower absolute zeta potential value of 50 nm PS-NPs induced the stronger interaction between PS-NPs and algae as compared to 70 nm PS-NPs, leading to severe membrane damage and the loss of esterase activity as well as settlement. These findings emphasized the importance of considering the impacts of commercial PS-NPs properties in toxicity evaluation.