The aim of this study was to examine the effect of positively charged (amine-modified) and negatively charged (carboxyl-modified) polystyrene nanoplastics (PS NPs) on the nanostructural, nanomechanical, and antioxidant responses of the marine diatom Cylindrotheca closterium. The results showed that both types of PS NPs, regardless of surface charge, significantly inhibited the growth of C. closterium during short-term exposure (3 and 4 days). However, longer exposure (14 days) to both PS NPs types did not significantly inhibit growth, which might be related to the detoxifying effect of the microalgal extracellular polymers (EPS) and the higher cell abundance per PS NPs concentration. The exposure of C. closterium to both types of PS NPs at concentrations above the corresponding concentrations that resulted in a 50% reduction of growth (EC50) demonstrated phytotoxic effects, mainly due to the excessive production of reactive oxygen species, resulting in increased oxidative damage to lipids and changes to antioxidant enzyme activities. Diatoms exposed to nanoplastics also showed a significant decrease in cell wall rigidity, which could make the cells more vulnerable. Atomic force microscopy images showed that positively charged PS NPs were mainly adsorbed on the cell surface, while both types of PS NPs were incorporated into the EPS that serves to protect the cells. Since microalgal EPS are an important food source for phytoplankton grazers and higher trophic levels, the incorporation of NPs into the EPS and interactions with the cell walls themselves may pose a major threat to marine microalgae and higher trophic levels and, consequently, to the health and stability of the marine ecosystem.