Silver nanoparticles (AgNPs) have found wide application in various commercial, medical and industrial products due to their unique properties. Their broad use has led to an increased release into aquatic environments, posing a threat to primary producers. Despite progress in research, the evaluation of AgNPs toxicity towards natural phytoplankton assemblages from different lake conditions is lacking. The present study investigates the toxicity of AgNPs on natural phytoplankton assemblages from lakes with contrasting trophic status. To this end, phytoplankton assemblages from mesotrophic and eutrophic lakes were exposed to 10 and 100 μg L⁻¹ of citrate-coated AgNPs for 48 h. Flow cytometry was used to characterise phytoplankton taxonomic groups and assessed physiological responses at the single cell level following AgNPs exposure. Results showed that phytoplankton assemblages from the eutrophic lake are more sensitive to AgNPs compared to those from the mesotrophic lake. The evaluation of phytoplankton sub-population also revealed that cyanobacteria were more sensitive to AgNPs than other eukaryotic phytoplankton regardless of trophic conditions. Moreover, small phytoplankton species were more affected than large phytoplankton species in both lakes. These findings highlight the need to consider both environmental context and assemblage composition when assessing AgNPs impacts on aquatic ecosystems.