The two major forms of cholinesterase enzymes found in the mammalian brain are acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). BuChE usually found mainly in glial cells and neuron in normal physiological condition, whereas AChE found near nerve synapse and axons, both are involved in the breakdown of acetylcholine (ACh) in the brain. The dual inhibition of these enzymes is considered as a promising strategy for the treatment of a neurological disorder such as Alzheimer’s disease, senile dementia, ataxia, and myasthenia gravis. The objective is to study the dual anticholinesterase activity of ajoene using in silico and in vitro methods. The anticholinesterase activity of ajoene was evaluated using Ellman’s assay, and molecular docking was performed on Schrödinger suite software. The present study demonstrated ajoene ([E, Z]‑4, 5, 9‑trithiadodeca‑1, 6, 11‑triene‑9‑oxide) inhibited both AChE and BuChE in a concentration‑dependent manner. The IC50 value of ajoene was 2.34 mM for AChE and 2.09 mM for BuChE. Kinetic studies showed mixed noncompetitive inhibition of AChE and uncompetitive inhibition of BuChE. Molecular docking studies revealed that ajoene interacts hydrophobically with catalytic residues of AChE while in case of BuChE the interaction is through noncatalytic binding site residues. Ajoene exhibits dual inhibitory activity against both AChE and BuChE enzymes.