The ecotoxicological effects of nanoplastic (NPs) and microplastic (MPs) polystyrene particles' (PS) on Hyalella azteca were studied in three tests designed to investigate various hypotheses and explore potential mechanisms of interaction between MPs, NPs and this species. The following materials were used: fluorescent nanoplastic nanoPS of 15–18 nm diameter, non-modified nanoPS 25 nm, and functionalized (aminometyl)polystyrene (PS-NH2). Short-term exposure of 7 and 14 days, and long-term exposure of 42 days, were conducted using three different types of PS at varying concentrations (0.01, 0.18, 1, 18, 180 mg L−1). The experiments were carried out through two methods: PS introduced via food and PS dispersed in the environment (referred to as the “medium”). The effects were comprehensively assessed by measuring the activity of selected oxidative stress biomarkers (acetylcholinesterase AChE, catalase CAT, and glutathione s-transferase GST), and monitoring parameters such as size, growth, reproduction rate, and the presence of possible malformations. The statistically significant effect was observed with PS-NH2 (37–74 μm) and fluorescent nanoPS (15–18 nm), whereas nanoPS of 25 nm were nearly inert. The discussion is focused on four observed aspects: (i) the impact of the surface characteristics and functional group modifications of PS particles on their overall effect on biota, (ii) the limitations of using a typical concentration parameter for tests comparison, with a proposal to adopt total surface area of MPs and NPs instead – reflecting the overall surface exposed to the environment, rather than solely relying on the mass or volume, (iii) the influence of feeding regimen (exposure at varying concentrations in food or medium compared to no exposure) on the ecotoxicological effect, and (iv) the potential of Hyalella azteca as a sentinel species for monitoring microplastic transport in both freshwater and brackish waters environments. Finally, the physical and chemical properties of all three PS types were characterized to better understand their mutual interaction with biota from the material perspective.