Three polyenylpyrone metabolites, pyridinopyrones A to C (1–3), have been isolated from the culture broth of a marine-derived Streptomyces sp., strain CNQ-301. The structures of the pyridinopyrones were assigned on the basis of chemical modification and combined spectroscopic methods, focusing on interpretation of 1D and 2D NMR data. Pyridinopyrones B and C (2, 3), examined as an inseparable mixture of methyl positional isomers, were ultimately defined by hydrogenation and NMR analysis of a saturated derivative. The biosynthesis of these metabolites was defined by the incorporation of stable isotope-labeled precursors, revealing that the biosynthetic starter unit is nicotinic acid, while the polyene chain and pendant methyl groups are acetate- and methionine-derived, respectively.