Paraoxonase 1 (PON1), an esterase linked to high-density lipoprotein (HDL), is known to have strong antioxidant and anti-cardiovascular properties. In this study, eleven acetohydrazide-based sulfonamide derivatives were synthesized. All compounds were characterized by appropriate spectroscopic techniques and elemental analyses. To better understand the inhibitory properties of the new sulfonamide derivatives, their in vitro effects on purified PON1 enzyme activity were studied. For this purpose, PON1 was purified from human serum using simple chromatographic methods. In the experimental study, the novel compounds were found to be potent inhibitors of paraoxonase 1. The mode of binding to the enzyme, for the relatively active compounds, was investigated through molecular docking. The most active compound N′-(naphthalen-2-ylsulfonyl)-2-(2-oxo-1,3-benzothiazol-3(2H)-yl)acetohydrazide demonstrated the highest interaction with the enzyme. Furthermore, the electrochemical properties of these compounds were assessed through DFT (density functional theory) analysis. N′-(naphthalen-2-ylsulfonyl)-2-(2-oxo-1,3-benzothiazol-3(2H)-yl)acetohydrazide is anticipated to have the highest potential to take part in electron exchanges whereas N′-[(4-fluorophenyl)sulfonyl]-2-(2-oxo-1,3-benzoxazol-3(2H)-yl)acetohydrazide is expected to have the highest chemical stability.