In this study, we investigated the interaction between arsenate (AsV) and phosphate (PO43–) in freshwater phytoplankton using single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS). This study aimed to elucidate the influence of varying PO43– concentrations on arsenic (As) uptake and distribution at the single-cell level, providing insights into intraspecies diversity. Two species of freshwater phytoplanktons, Scenedesmus acutus and Pediastrum duplex, were cultured under different concentrations of PO43– and AsV in a controlled laboratory environment. Scenedesmus acutus, a species with strong salt tolerance, and Pediastrum duplex, known for its weak salt tolerance, were selected based on their contrasting behaviors in previous studies. SC-ICP-MS revealed non-uniform uptake of As by individual phytoplankton cells, with distinct variations in response to PO43– availability. Arsenic uptake by both species declined with a high PO43– level after 7 days of exposure. However, after 14 days, As uptake increased in S. acutus with higher PO43– concentrations, but decreased in P. duplex. Moreover, our findings revealed differences in cell morphology and membrane integrity between the two species in response to AsV and various PO43– concentrations. S. acutus maintained cell integrity under all experimental culture conditions, whereas P. duplex experienced cell lysis at elevated AsV and PO43– concentrations. This study highlights the varying responses of freshwater phytoplankton to changes in AsV and PO43– levels and underscores the advantages of SC-ICP-MS over conventional ICP-MS in providing detailed, cellular level insights. These findings are crucial for understanding and managing As pollution in aquatic ecosystems.