Picocyanobacteria are widespread and globally significant primary producers. In brackish waters, picocyanobacterial populations are composed of diverse species with both single-cell and colony-forming lifestyles. Compared to their marine counterparts, brackish picocyanobacteria are less well characterized and the focus of research has been weighted toward single-cell picocyanobacteria. Here, we investigate the uptake dynamics of single and colony-forming picocyanobacteria using incubations with dual carbon-13 and inorganic (ammonium and nitrate) or organic (urea and amino acids) nitrogen-15 sources during August and September 2020 in the central Baltic Sea. Phytoplankton community and group-specific uptake rates were obtained using an elemental analyzer isotope ratio mass spectrometer (EA-IRMS) and nano secondary-ion mass spectrometry (NanoSIMS). Picocyanobacteria contributed greater than one third of the ammonium, urea, amino acids, and inorganic carbon community uptake/fixation in September but < 10% in August when phytoplankton biomass was higher. Overall, single-cell ammonium and urea uptake rates were significantly higher for single-celled compared to colonial picocyanobacteria. In a 6-yr offshore central Baltic Sea time series (2015–2020), summer abundances of colonial picocyanobacteria reached up to 10⁵ cells mL⁻¹ and represented > 5% of the average phytoplankton biomass, suggesting that they are periodically important for the ecosystem. Colonial strain identification was not distinguishable using 16S rRNA gene amplicon data, highlighting a need for refined tools for identification of colonial forms. This study shows the significance of single-celled brackish picocyanobacteria to nutrient cycling and the importance of considering uptake and lifestyle strategies when assessing the role of picocyanobacteria in aquatic ecosystems. © 2024 The Author(s). Limnology and Oceanography published by Wiley Periodicals LLC on behalf of Association for the Sciences of Limnology and Oceanography.