Summary: Despite the well-established medicinal values of Jimson weed (Datura stramonium L.), this medicinal plant has been associated with neurological effects such as hallucination and anxiety in folklore. This study examined the effect of alkaloid extracts from the leaf and fruit of Jimson weed on critical enzymes of the monoaminergic [monoamine oxidase (MAO)] and cholinergic [acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)] systems of neurotransmission. Alkaloid extracts were prepared by solvent extraction method and their interaction with the activities of MAO, AChE, and BChE were assessed (in vitro). Gas chromatography coupled with mass spectroscopic (GC-MS) characterization of the extracts was also carried out. The results revealed that the extracts inhibited the activity of the enzymes assayed for in a concentration-dependent manner. Considering the IC50 values, the fruit extract had more potent (P < 0.05) inhibitory effect on the enzymes’ activities, compared to the leaf extract. GC-MS characterization revealed the presence of atropine, scopolamine, amphetamine, 3-methoxyamphetamine, 3-ethoxyamhetamine cathine, spermine, phenylephrine, and 3-piperidinemethanol, among others in the extracts. The alteration of activities of these critical enzymes of the cholinergic and monoaminergic signaling may be responsible for the reported neurological effects of this medicinal plant in folklore; nevertheless, the fruit extract exhibited more neuromodulatory effect than the leaf.