In this study, we propose identifying potential novel compounds targeting carbonic anhydrase I and II. Herein, we have designed and synthesized new benzimidazole-hydrazide-hydrazones derivatives (4a-4r) to investigate the effects of these synthesized compounds on CA isoenzymes. The compounds' 1H NMR, 13C NMR, and HRMS spectra were used to confirm their chemical structures. The synthetic derivatives were screened for their inhibitory potential against carbonic anhydrase I and II by in vitro assay. These compounds have IC50 values of 3.727–1.493 μM (hCA I) and 3.892–1.547 μM (hCA II). Inhibition types and Ki values of the compounds were determined. The Ki values of the compounds were 3.006 ± 0.17 μM-0.356 ± 0.0 μM (hCA I) and 2.923 ± 0.15 μM-0.346 ± 0.0 μM (hCA II). Acetazolamide (AAZ) was used as the reference in the study. The most potent derivatives, a 4-methoxy derivative (compound 4k) and 4-(trifluoromethyl) derivative (compound 4g), than AAZ (IC50 = 2.26 μM) and their IC50 values were found as 1.493 μM and 1.675 μM, respectively. Compared to AAZ, the other derivatives having more effect on hCA I were compounds 4b, 4e, 4l, 4m, 4n, and 4o. The compounds gave IC50 values of 1.743, 1.789, 1.933, 1.966, 1.983, and 1.986 μM, respectively. Compounds 4a-4r found no more effective inhibitory activity against hCA II isozyme than AAZ (IC50 = 1.17 μM). According to the in vitro test results, detailed protein-ligand interactions of the compounds 4b and 4k exhibited considerably low binding energies, suggesting strong interaction affinities against hCA I. In addition, the cytotoxic effects of the compounds on the L929 healthy cell line were evaluated.