In five steps, new compounds 5a, 5b of thiadiazole-hydrazone derivatives were synthesized. Various spectral methods, such as 1H NMR, 13C NMR, and elemental analyses, were used to clarify the structures of the compounds. Three cancer cell lines (MCF7, MDA, and HT-29) and one healthy cell line (L929) were tested for the cytotoxicity activity of synthetic compounds, as well as their inhibitory action against carbonic anhydrase I and II isoenzymes (hCA I and hCA II). Among them, the compound 5b exhibited remarkable CA inhibitory activities compared to a standard inhibitor with IC50 values at of 27 µM for hCA I and 33,46 µM for hCA II. The compounds have been found to be ineffective against cancer cell lines. Furthermore, the compounds were found to be non-toxic to the healthy cell line.