The absolute configuration of the polyketide natural product (-)-enterocin was established by two independent approaches. In the first approach, synthetic enterocin with a defined configuration was compared to the natural product. While identical in all scalar properties, the compound displayed an opposite specific rotation and a different chiral HPLC retention time when compared with (-)-enterocin. In a second approach, the vibrational circular dichroism (VCD) of the natural product was measured and shown to be opposite to the calculated VCD of its enantiomer.