The discovery of novel and unique target−drug pairs for the treatment of human diseases such as cancer and bacterial infections is an urgent goal of chemical and pharmaceutical sciences. Natural products represent an inspiring source of compounds for designing chemical biology methods with applications in target identification and characterization. Inspired by the huge structural diversity of γ-butyrolactones, which constitute up to 10% of all known compounds of natural origin, we extended the “activity-based protein profiling” (ABPP) target identification technology to this promising and so far unexplored natural compound class. We designed and synthesized a comprehensive set of natural product-derived γ-lactones and thiolactones that varied in protein reactivity. Several important bacterial enzymes that are involved in diverse cellular functions such as metabolism (dihydrolipoyl dehydrogenase and 6-phosphofructokinase), cell wall biosynthesis (MurA1 and MurA2), and protein folding (trigger factors) were obtained. Especially protein folding in bacteria could represent a novel strategy for antibiotic intervention and requires chemical tools for characterization and inhibition. Future studies that extend structural modifications to protein reactive α-methylene-γ-butyrolactone as well as to reversible binding γ-lactones and thiolactones will reveal if this premise holds true.