Freshwater picocyanobacteria (Syn/Pro clade) contribute substantially to the primary production of inland waters, especially when nitrogen is limiting or co-limiting. Nevertheless, they remain poorly understood ecologically and genomically, with research on their nitrogen acquisition mainly focused on inorganic sources. However, dissolved organic nitrogen is often a major component of the freshwater nitrogen pool and it is increasingly evident that many forms are bioavailable. Comparative genomic analyses, axenic growth assays, and proteomic analyses were used here to investigate organic nitrogen acquisition mechanisms in the Syn/Pro clade. Comparative analysis of the genomes of 295 freshwater and marine strains of picocyanobacteria identified a large diversity of amino acid transporters, the absence of degradation pathways for five amino acids (asparagine, phenylalanine, serine, tryptophan, and tyrosine), and alternative mechanisms for chitin assimilation (direct chitin catabolise vs initial acetylation to chitosan and subsequent degradation). Growth assays demonstrated the widespread bioavailability of amino acids, including basic amino acids though the known basic amino acid transporter is not encoded. This suggests further genetic components are involved, either through extracellular catabolism or the presence of novel transporters. Proteomic analysis demonstrates the dual utilisation of nitrogen and carbon from the amino acid substrate and provides evidence for a mild stress response through the up-regulation of lysine biosynthesis and FtsH1, potentially caused by accumulation of secondary metabolites. Our results are relevant to understanding how picocyanobacteria have come to thrive in dissolved organic nitrogen-rich oligotrophic environments and explores how their different molecular capabilities may influence communities between habitats.