<jats:title>Summary</jats:title><jats:p> <jats:italic>Nodularia spumigena</jats:italic> is a filamentous nitrogen‐fixing cyanobacterium that forms toxic blooms in brackish water bodies worldwide. Spumigins are serine protease inhibitors reported from a single strain of <jats:italic>N. spumigena</jats:italic> isolated from the Baltic Sea. These linear tetrapeptides contain non‐proteinogenic amino acids including a C‐terminal alcohol derivative of arginine. However, very little is known about these compounds despite the ecological importance of <jats:italic>N. spumigena</jats:italic>. We show that spumigins are assembled by two non‐ribosomal peptide synthetases encoded in a 21 kb biosynthetic gene cluster. The compact non‐ribosomal peptide synthetase features a reductive loading and release mechanism. Our analyses demonstrate that the bulk of spumigins produced by <jats:italic>N. spumigena</jats:italic> are released as peptide aldehydes in contrast to earlier findings. The main spumigin E variant contains an argininal residue and is a potent trypsin inhibitor. Spumigins were present in all of the <jats:italic>N. spumigena</jats:italic> strains isolated from the Baltic Sea and comprised up to 1% of the dry weight of the cyanobacterium. Our results demonstrate that bloom‐forming <jats:italic>N. spumigena</jats:italic> strains produce a cocktail of enzyme inhibitors, which may explain in part the ecological success of this cyanobacterium in brackish water bodies worldwide.