Updated project metadata. Usnic acid is a natural product with versatile biological activities against various organisms. We here utilize a chemical proteomic strategy to gain insights into its target scope in bacterial and human cells. First, we excluded DNA binding as a major reason for its antibacterial activity and second commenced with target profiling which unravelled several metal cofactor-dependent enzymes in both species. This is in line with the metal complexation activities of usnic acid and suggests a polypharmacological mode of action. Interestingly, our synthetic studies revealed a selectivity switch at usnic acid which maintains antibacterial activity but lacks strong cytotoxic effects.