Two new alpha-pyridone metabolites, iromycins E and F, were isolated from cultures of strain Streptomyces sp. Dra 17, thus expanding the recently discovered iromycin family. The inhibitory potential on the mitochondrial respiratory chain was examined and revealed that iromycin metabolites block NADH oxidation in beef heart submitochondrial particles with different efficacy, yet remarkably show only very low cytotoxicity. Difference spectroscopic studies indicated that iromycins inhibit the electron transport at the site of complex I (NADH-ubiquinone oxidoreductase). Derivatives of the natural products were semisynthetically prepared and provided detailed insights into structure-activity relationships. Drawn from these results, there are strong similarities with the piericidins, which are among the most potent complex I inhibitors of the mitochondrial electron transport chain. Furthermore, total synthesis afforded new analogues, and the non-natural iromycin S (IC50 = 58 ng/mL) emerged as the most active compound, thus opening avenues of future studies with the iromycins as new valuable biochemical tools.