Nanoplastics persist in aquatic environments and can adsorb a wide range of organic and inorganic contaminants owing to their small size and high specific surface area. Consequently, they function not only as particulate pollutants but also as vectors that influence the transport, bioavailability, and toxicity of coexisting contaminants. Despite these interactions, most previous studies have evaluated nanoplastic toxicity under simplified laboratory conditions using standard test media, which do not adequately represent natural aquatic environments. In this study, we investigated the ecotoxicological effects of polystyrene nanoplastics (PS-NPs) under environmentally relevant freshwater conditions. Freshwater collected from an urban stream was used as the exposure medium, and Daphnia magna served as the model organism. Exposure to PS-NPs in freshwater altered the physicochemical characteristics of the particles, resulting in distinct toxicological responses compared with those observed in the standard medium. These responses included changes in growth, reproduction, gut microbiota composition, and biochemical and molecular biomarkers in D. magna. Our findings demonstrate that interactions between nanoplastics and naturally occurring freshwater constituents can substantially modify nanoplastic behavior and toxicity, highlighting the importance of incorporating environmentally realistic exposure conditions into ecological risk assessments of nanoplastics.