Background: The carbonic anhydrases (CAs) which are found in most living organisms is a member of the zinc-containing metalloenzyme family. The abnormal levels and activities are frequently associated with various diseases therefore CAs have become an attractive target for the design of inhibitors or activators that can be used in the treatment of those diseases. Methods: Herein, we have designed and synthesized new benzimidazole-hydrazone derivatives to investigate the effects of these synthesized compounds on CA isoenzymes. Chemical structures of synthesized compounds were confirmed by1H NMR,13C NMR, and HRMS. The synthetic derivatives were screened for their inhibitory potential against carbonic anhydrase I and II by in vitro assay. Results: These compounds have IC50 values of 5.156-1.684 µM (hCA I) and 4.334-2.188 µM (hCA II). Inhibition types and Ki values of the compounds were determined. The Ki values of the compounds were 5.44 ± 0.14 µM-0.299 ± 0.01 µM (hCA I) and 3.699 ± 0.041 µM-1.507 ± 0.01 µM (hCA II). The synthetic compounds displayed inhibitory action comparable to that of the clinically utilized reference substance, acetazolamide. According to this, compound 3p was the most effective molecule with an IC50 value of 1.684 µM. Accordingly, the type of inhibition was noncompetitive and the Ki value was 0.299 ± 0.01 µM. Conclusion: According to the in vitro test results, detailed protein-ligand interactions of the compound 3p, which is more active against hCA I than standard azithromycin (AZM), were analyzed. In addition, the cytotoxic effects of the compounds on the L929 healthy cell line were evaluated.