In this study, first, Schiff base-containing chalcone derivatives were synthesized. The human carbonic anhydrase (hCA) isoenzymes I and II were then purified from human erythrocytes using Sepharose-4B-l-tyrosine-sulfanilamide affinity chromatography. In addition, the inhibitory effects of the newly synthesized compounds on the activities of hCA and acetylcholinesterase (AChE) were investigated in vitro, using the esterase and acetylcholine iodide method. The IC(50)values were determined and theK(i)values of AChE and hCA activities were calculated from the Lineweaver-Burk graphs determined in this study. The hCA I isoform was inhibited by these chalcone derivatives containing Schiff bases (3a-jand5a-f) in low nanomolar levels, whoseK(i)values ranged between 141.88 +/- 24.10 and 2,234.47 +/- 38.11 nM. Against the physiologically dominant isoform hCA II, the compounds demonstratedK(i)values varying from 199.31 +/- 40.45 to 602.79 +/- 263.22 nM. Also, these compounds effectively inhibited AChE, withK(i)values ranging from 20.41 +/- 6.04 to 125.94 +/- 23.88 nM. According to these results, the newly synthesized molecules were found to be potent inhibitors of these enzymes.