Background Thiophene(s) are an important group in therapeutic applications, and sulfonamides are the most important class of carbonic anhydrase (CA) inhibitors. In this study, inhibition effects of some thiophene-based sulfonamides on human erythrocytes carbonic anhydrase I and II isoenzymes (hCA-I and hCA-II) were investigated. Thiophene-based sulfonamides used in this study showed potent inhibition effect on both isoenzymes at very small concentrations. Materials and methods We report on the purification of the carbonic anhydrase I and II isoenzymes (hCA-I and hCA-II) using affinity chromatography method. The inhibition effect of the thiophene-based sulfonamides was determined by IC(50)and K(i)parameters. A molecular docking study was performed for each molecule. Results Thiophene-based sulfonamides showed IC(50)values of in the range of 69 nM to 70 mu M against hCA-I, 23.4 nM to 1.405 mu M against hCA-II. K(i)values were in the range of 66.49 +/- 17.15 nM to 234.99 +/- 15.44 mu M against hCA-I, 74.88 +/- 20.65 nM to 38.04 +/- 12.97 mu M against hCA-II. Thiophene-based sulfonamides studied in this research showed noncompetitive inhibitory properties on both isoenzymes. To elucidate the mechanism of inhibition, a molecular docking study was performed for molecules 1 and 4 exhibiting a strong inhibitory effect on hCA-I and hCA-II. The compounds inhibit the enzymes by interacting out of catalytic active site. The sulfonamide and thiophene moiety played a significant role in the inhibition of the enzymes. Conclusion We hope that this study will contribute to the design of novel thiophene-based sulfonamide derived therapeutic agents that may be carbonic anhydrase inhibitors in inhibitor design studies.