<jats:title>Abstract</jats:title><jats:p>Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, sphaerocyclamide (1), from <jats:italic>Sphaerospermopsis</jats:italic> sp. LEGE 00249. The sphaerocyclamide biosynthetic gene cluster (<jats:italic>sph</jats:italic>) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the <jats:italic>sph</jats:italic> gene cluster in <jats:italic>Escherichia coli</jats:italic> confirmed the connection between genomic and mass spectrometric data. Unambiguous establishment of the orientation and site of prenylation required the full structural elucidation of 1 using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound 1 was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium <jats:italic>Halomonas aquamarina</jats:italic> CECT 5000.