A dual-tail approach was applied to the design of a novel series of 2-thiopyrimidine-benzenesulfonamides as carbonic anhydrase (CA) inhibitors. The design strategy is based on the hybridization between a benzenesulfonamide moiety as Zn<sup>2+</sup> binding group and 2,4-disubstituted thiopyridimidine as a tail. Among the synthesized compounds, 14h displayed the highest potency (K<sub>i</sub> = 1.72 nM) and selectivity for CA II over the isoforms CA IX and CA XII with selectivity indexes of 50 and 5.26, respectively. Meanwhile, compounds 14a and 14l displayed a potent inhibitory activity against CA IX (K<sub>i</sub> = 7.4 and 7.0 nM, respectively) compared with the reference drug acetazolamide (AAZ) (K<sub>i</sub> = 25 nM), and compound 14l showed higher potency (K<sub>i</sub> = 4.67 nM) than AAZ (K<sub>i</sub> = 5.7 nM) against the tumor-associated isoform CA XII. Evaluation of the antiproliferative activity in NCI single-dose testing of selected hybrids revealed a pronounced potency of the selective CA II inhibitor 14h against most of the tested NCI cancer cell lines. Moreover, compound 14h demonstrated an IC<sub>50</sub> values ranging from 2.40 to 4.50 μM against MCF-7, T-47D, MDA-MB-231, HCT-116, HT29 and SW-620. These results demonstrate that CA II inhibition can be an alternative therapeutic target for cancer treatment. A cell cycle analysis of MCF-7 and MDA-MB-231 showed that treatment with 14h arrested both cell lines at the G2/M phase with significant accumulation of cells in the pre-G1 phase. Moreover, compound 14h showed a noticeable induction of late apoptosis and necrotic cell death of both cell lines compared with untreated cells as a control. A molecular docking study suggested that the sulfonamide moiety accommodates deeply in the CA active site and interacts with the Zn<sup>2+</sup> ion while the dual-tail extension interacts with the surrounding amino acids via several hydrophilic and hydrophobic interactions, which affects the potency and selectivity of the hybrids.