Mycolactones are polyketide toxins produced by Mycobacterium ulcerans, the causative agent of the tropical skin disease known as Buruli ulcer. Development of novel therapeutic agents from mycolactones has been hindered by the difficulty of producing sufficient amounts of material. Here, we describe the successful adaptation of M. ulcerans to suspension cultivation and the development of a fed-batch fermentation process that was scaled up to 150 l. In addition to producing mycolactones A and B, a number of new mycolactone-related compounds were also observed.