While bacterial genomes typically contain numerous secondary metabolite biosynthetic gene clusters, only a small fraction of these are expressed at any given time. The remaining majority is inactive or silent, and methods that awaken them would greatly expand our repertoire of bioactive molecules. We recently devised a new approach for identifying inducers of silent gene clusters and proposed that the clinical antibiotic trimethoprim acted as a global activator of secondary metabolism in Burkholderia thailandensis. Herein, we report that trimethoprim triggers the production of over 100 compounds that are not observed under standard growth conditions, thus drastically modulating the secondary metabolic output of B. thailandensis. Using MS/MS networking and NMR, we assign structures to ∼40 compounds, including a group of new molecules, which we call acybolins. With methods at hand for activation of silent gene clusters and rapid identification of small molecules, the hidden secondary metabolomes of bacteria can be interrogated.