The proposed structure 9 of mycolactone F, isolated from the fish pathogen Mycobacterium marinum, was synthesized from the core alcohol 7 and unsaturated acid 6. The H-1 NMR spectrum of synthetic 9 matched well the reported 1 H NMR spectrum of natural mycolactone F, thereby demonstrating the structure of mycolactone F to be 9 or its remote diastereomer 10. In order to develop an analytical method to distinguish them, 10 was synthesized from 7 and the antipode of 6. With the use of synthetic materials, a chiral HPLC method was developed to differentiate 10 from 9. This analytical method was then applied to the natural product, thereby establishing that the complete structure of OH OH mycolactone F surprisingly is 10, rather than 9. Intriguingly, the absolute configuration of the unsaturated fatty acid of mycolactone F from the fish pathogen M. marinum corresponds to the antipode of that present in natural mycolactones A/B and C from the human pathogen M. ulcerans.