Objective: In this study, novel 5-fluoroindolylmethylene hydrazone derivatives were synthesized and assessed for their potential inhibitory effects on human carbonic anhydrase I and II (hCA I and II) enzymes through both in vitro and in silico approaches. Material and Method: Accordingly, starting from 5-fluoroindole, 5-fluoroindole-3-carboxaldehyde was synthesized. The target final compounds were then obtained by condensing substituted phenylhydrazine with 5-fluoroindole-3-carboxaldehyde, resulting in three Schiff base derivatives (2a, 2b, and 2c). Result and Discussion: The synthesized compounds demonstrated effective inhibition of cytosolic carbonic anhydrase isoforms hCA I and II, with Ki values ranging from 32.28±7.09 to 74.86±10.90 nM for hCA I and 8.79±1.92 to 60.40±14.64 nM for hCA II. Among them, compound 2a exhibited the most potent inhibitory effect on both isoenzymes. In vitro results were verified with the results obtained by docking studies and interactions with enzymes were demonstrated. These novel 5-fluoroindolylmethylene hydrazone derivatives show promise as potent inhibitors of cytosolic CA isoenzymes.