Incorporation of the purine/pyrimidine moieties as tails to classical benzenesulfonamide scaffolds afforded two series of human (h) carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The compounds were designed according to the molecular hybridization approach, in order to modulate the interaction with different CA isozymes and exploit the antitumor effect of uracil and adenine derivatives in parallel and synergic mode to the inhibition of the tumor-associated hCA IX. The sulfonamides were investigated as inhibitors of four isoforms, cytosolic hCA I/II and transmembrane hCA IV/IX. The inhibitory profiles were dependent on the length and positioning of the spacer connecting the two pharmacophores. X-ray crystallography demonstrated the binding mode of an inhibitor to hCA II and hCA IX-mimic. Compounds endowed with the best hCA IX inhibitory efficacy were evaluated for antiproliferative activity against HT-29 colon cancer cell lines. The in vitro results suggest multiple mechanisms of action are responsible for the compounds' cytotoxic efficacy.