Strain SpD081030SC-03, representing a novel species of Streptomyces, was isolated from a marine sponge. Two 3,5,6-trisubstituted 2(1H)-pyrazinones, JBIR-56 (1) and JBIR-57 (2), were isolated from a culture of SpD081030SC-03. The planar structures of 1 and 2 were assigned on the basis of extensive NMR and MS analyses. In addition, analyses of the methylated derivative of 1 confirmed a 3,5,6-trisubstituted 2(1H)-pyrazinone moiety. The absolute configurations of the amino acid residues were determined by application of Marfey's method. Because 1 did not appear to comprise the normal connection of amino acid units, we confirmed its structure by the total synthesis of 1. Biosynthetically, 1 consists of a unique skeleton connected to the peptide chain at C-5 of the pyrazinone ring.