Human carbonic anhydrase I and II isoenzymes (hCA I and II) and acetylcholinesterase (AChE) are important metabolic enzymes that are closely associated with various physiological and pathological processes. In this study, we investigated the inhibition effects of some sulfonamides on hCA I, hCA II, and AChE enzymes. Both hCA isoenzymes were purified by Sepharose-4B-L-Tyrosine-5-amino-2-methylbenzenesulfonamide affinity column chromatography with 1393.44 and 1223.09-folds, respectively. Also, some inhibition parameters including IC50 and K-i values were determined. Sulfonamide compounds showed IC50 values of in the range of 55.14 to 562.62 nM against hCA I, 55.99 to 261.96 nM against hCA II, and 98.65 to 283.31 nM against AChE. K-i values were in the range of 23.40 +/- 9.10 to 365.35 +/- 24.42 nM against hCA I, 45.87 +/- 5.04 to 230.08 +/- 92.23 nM against hCA II, and 16.00 +/- 45.53 to 157.00 +/- 4.02 nM against AChE. As a result, sulfonamides had potent inhibition effects on these enzymes. Therefore, we believe that these results may contribute to the development of new drugs particularly in the treatment of some disorders.