<jats:p>New isolates of Streptomyces champavatii were isolated from marine sediments of the Gotland Deep (Baltic Sea), from the Urania Basin (Eastern Mediterranean), and from the Kiel Bight (Baltic Sea). The isolates produced several oligopeptidic secondary metabolites, including the new octapeptide champacyclin (1a) present in all three strains. Herein, we report on the isolation, structure elucidation and determination of the absolute stereochemistry of this isoleucine/leucine (Ile/Leu = Xle) rich cyclic octapeptide champacyclin (1a). As 2D nuclear magnetic resonance (NMR) spectroscopy could not fully resolve the structure of (1a), additional information on sequence and configuration of stereocenters were obtained by a combination of multi stage mass spectrometry (MSn) studies, amino acid analysis, partial hydrolysis and subsequent enantiomer analytics with gas chromatography positive chmical ionization/electron impact mass spectrometry (GC-PCI/EI-MS) supported by comparison to reference dipeptides. Proof of the head-to-tail cyclization of (1a) was accomplished by solid phase peptide synthesis (SPPS) compared to an alternatively side chain cyclized derivative (2). Champacyclin (1a) is likely synthesized by a non-ribosomal peptide synthetase (NRPS), because of its high content of (d)-amino acids. The compound (1a) showed antimicrobial activity against the phytopathogen Erwinia amylovora causing the fire blight disease of certain plants.