Silver nanoparticles (AgNPs) are widely used nanomaterials, and understanding their effects on phytoplankton is essential for assessing ecological risks. While AgNP toxicity has been extensively studied in single species, the role of interspecies interactions on toxicity outcomes remains poorly understood. This study investigates the effects of AgNPs and Ag⁺ on phytoplankton, focusing on species-specific responses and interspecies interactions within artificial assemblages. Monocultures and assemblages of Chlamydomonas reinhardtii, Cyclotella meneghiniana, and Synechocystis sp. were exposed to AgNPs (1-3000 µg L^-1 ) and Ag⁺ (0.05-300 µg L^-1 ) to assess growth inhibition, photosynthetic efficiency, and pigment fluorescence. Spectral flow cytometry enabled high-resolution, single-cell monitoring of cell abundance and pigment fluorescence in the assemblages. Results revealed species-specific toxicity, with Synechocystis sp. being most affected by AgNPs, while C. meneghiniana showed greater tolerance. Ag⁺ exposure triggered rapid effects, including reduced photosynthetic efficiency within 2 h, whereas AgNPs caused delayed but persistent toxicity. Experiments with artificial assemblages revealed that interspecies interactions modulated silver toxicity. Co-occurrence of species alleviated AgNP stress, resulting in significantly higher 72h-EC₅₀ for some species and sustained photosynthetic performance in the cohabiting eukaryotes. These findings demonstrated that phytoplankton assemblages' composition strongly influences AgNP toxicity and highlighted spectral flow cytometry as a powerful tool for resolving species-specific responses within complex assemblages. The study emphasized the importance of integrative, assemblage-based approaches in ecotoxicology to more accurately predict ecosystem-level impacts of pollutants.