A series of coumarins and the corresponding 2-thioxocoumarines were prepared and tested for their inhibition profiles against four physiologically relevant human carbonic anhydrases (hCAs, EC 4.2.1.1), isoforms hCA I, II, IX, and XII. The X-ray crystal structure of 6-hydroxy-2-thioxocoumarin bound to hCA II revealed an unprecedented and unexpected inhibition mechanism for this new class of inhibitors when compared to isostructural coumarins. Unlike coumarins which are hydrolyzed by the esterase CA activity to the corresponding 2-hydroxy-cinnamic acid derivatives, the 2-thioxocoumarin was observed intact when bound to hCA II, with its exo-sulfur atom anchored to the zinc-coordinated water molecule, whereas the scaffold establishing favorable contacts with amino acid residues from the active site. This inhibition mechanism is very different from the one observed for hydrolyzed coumarins, which occlude the entrance of the active site cavity. This versatility in the binding mode of coumarins/thioxocoumarins has important consequences for the design of isoform-selective CA inhibitors, some of which are in clinical use or clinical development for various pathologies, among which glaucoma, edema, epilepsy, neuropathic pain, and hypoxic tumors.