AbstractThis report presents the synthesis and characterization of a range of benzimidazolium salts featuring 3‐cyanopropyl groups on the 1^st nitrogen atom and varied alkyl groups on the 3^rd nitrogen atom within the benzimidazole structure. Benzimidazolium salts were synthesized by N‐alkylation of 1‐alkyl benzimidazole with 3‐cyanopropyl‐bromide. The new salts were characterized by ¹H and ¹³C‐NMR, FT‐IR spectroscopic and elemental analysis techniques. In this study, the enzyme inhibition abilities of seven nitrile substituted benzimidazolium salts were investigated against acetylcholinesterase (AChE) and carbonic anhydrase isoenzymes I and II (hCA I and hCA II). They showed a highly potent inhibition effect on AChE, hCA I and hCA II (K_i values are in the range of 26.71–119.09 nM for AChE, 19.77 to 133.68 nM for hCA I and 13.09 to 266.38 nM for hCA II). Reflecting the binding mode of the synthesized cyanopropyl series, the importance of the 2,3,5,6‐tetramethylbenzyl, 3‐methylbenzyl and 3‐benzyl groups for optimal interactions with target proteins, evaluated by molecular docking studies. At the same time, the docking findings support the inhibition constants (K_i) values of the related compounds in this study. Potential compounds were also evaluated by their pharmacokinetic properties were predicted.