The stereochmical determination of the potent respiratory chain inhibitors ajudazols A and B and the total synthesis of ajudazol B are reported. Configurational assignment was exclusively based on biosynthetic gene cluster analysis of both ketoreductase domains for hydroxyl-bearing stereocenters and one of the first predictive enoylreductase alignments for methyl-bearing stereocenters. The expedient total synthesis resulting in unambiguous proof of the predicted stereochemistry involves a short stereoselective approach to the challenging isochromanone stereotriad by an innovative asymmetric ortholithiation strategy a modular oxazole formation; and a late stage Z,Z-selective. Suzuki coupling.