Efforts to isolate carrier protein-mediated synthases from natural product-producing organisms using reporter-linked post-translational modification have been complicated by the efficiency of the endogenous process. To address this issue, we chose to target endogenous phosphopantetheinyl transferases (PPTases) for inhibitor design to facilitate natural product synthase isolation through a chemical genetics approach. Herein we validate secondary metabolism-associated PPTase for chemical probe development. We synthesized and evaluated a panel of compounds based on the anthranilate 4H-oxazol-5-one pharmacophore previously described to attenuate PPTase activity within bacterial cultures. Through the use of a new high-throughput Förster resonance energy transfer assay, we demonstrated that these compounds exclusively inhibit fatty acid synthase-specific PPTases. In vivo, a lead compound within this panel demonstrated selective antibiotic activity in a Bacillus subtilis model. Further evaluation demonstrated that the compound enhances actinorhodin production in Streptomyces coelicolor, revealing the ability of this class of molecules to stimulate precocious secondary metabolite production.