Paraoxonase 1 (PON1) was purified 148.80-fold in 37.92% yield by hydrophobic interaction chromatography technique. The purity of PON1 was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a single band of 43 kDa. The in vitro effects of nine different calcium channel blockers on PON1 activity were evaluated. All drugs strongly decreased PON1 activity, and IC50 levels were between 13.987 ± 0.59 and 238.104 ± 2.14 μM, Ki values between 8.58 ± 0.36 and 111 ± 1.27 μM. The drugs with the strongest inhibitory effect were nisoldipine with 13.987 ± 0.59 μM and nicardipine with 20.158 ± 0.43 μM. The mechanism of action for the inhibition of the enzyme by nisoldipine and nicardipine was investigated through molecular docking. The stability of enzyme–ligand complexes obtained from the docking was explored through molecular dynamics simulation. The binding affinity of the ligands toward the enzyme was also investigated through MMPBSA (molecular mechanics Poisson–Boltzmann surface area method). The computational analysis demonstrated these compounds could inhibit the enzyme. Nisoldipine had the strongest binding, and its complex was the most stable one. Furthermore, nicardipine was found to have the highest affinity toward the enzyme.