Only a few natural products possessing a diazeniumdiolate have been isolated and usually these compounds display a broad range of biological activities. Only recently the first diazeniumdiolate natural product biosynthetic gene cluster was identified in Burkholderia cenocepacia H111, which produces the fungicide (-)-fragin and the signal molecule (rac)-valdiazen. In this study, L-valine was identified as the initial substrate of (-)-fragin biosynthesis by feeding experiments using isotopically labeled amino acids. The formation of the diazeniumdiolate was chemically studied by several proposed intermediates. Our results indicate that the functional group is formed during an early stage of the biosynthesis. Furthermore, an oxime compound was identified as a degradation product of (-)-fragin and was also observed in the crude extract of the wild type strain. Moreover, structure-activity relationship analysis revealed that each moiety of (-)-fragin is essential for its biological activity.