Photochemistry of mycolactone A/B and related unsaturated fatty acid esters is reported. On exposure to visible light, mycolactone A/B gave a mixture of four photomycolactones. Pentaenoates and tetraenoates, representing the unsaturated fatty acid portion of mycolactone A/B, were found to show the reactivity profile parallel with that of mycolactone A/B. The structure of the four photomycolactones was elucidated via (1) structure determination of the four photoproducts in the tetraenoate series; (2) their transformation to the photoproducts in the pentaenoate and then mycolactone series. Triplet quenchers did not affect the photochemical transformation, thereby indicating an event at the singlet state. A concerted, photochemically allowed [4πs + 2πa] cycloaddition was suggested to account for the observed result. This study provided the structurally defined and homogeneous material, which allowed demonstration that photomycolactones exhibit significantly reduced cytotoxicity, compared with mycolactone A/B.