We report a one-pot procedure for the synthesis of asymmetrical ureido-containing benzenesulfonamides based on in situ generation of the corresponding isocyanatobenezenesulfonamide species, which were trapped with the appropriate amines. A library of new compounds was generated and evaluated in vitro for their inhibition properties against a representative panel of the human (h) metalloenzymes carbonic anhydrases (EC 4.2.1.1), and the best performing compounds on the isozyme II (i.e., <b>7c</b>, <b>9c</b>, <b>11g</b>, and <b>12c</b>) were screened for their ability to reduce the intraocular pressure in glaucomatous rabbits. In addition, the binding modes of <b>7c</b>, <b>11f</b>, and <b>11g</b> were assessed by means of X-ray crystallography.