The caseinolytic protease is a highly conserved serine protease, seminal in prokaryotic and eukaryotic protein homeostasis and regulatory proteolysis, and a promising antibacterial and anticancer drug target. Here, we describe the cystargolides as potent and the first natural β-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic genes in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show covalent inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semi-synthetic derivatives with improved cell permeability allowed us to confirm ClpP as a specific target within intact S. aureus cells and to demonstrate anti-virulence activity. In Streptomycetes griseus growth inhibition occurs. Crystal structures show cystargolide A covalently bound to all 14 active sites of S. aureus ClpP. Although synthetic β-lactones are known as the pioneering group of ClpP inhibitors, their co-crystallisation has not been successful, so far. The cystargolides now reveal the molecular mechanism of ClpP inhibition by β-lactone inhibitors.