Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood–brain barrier, and has demonstrated excellent safety and tolerability properties in clinical research. These characteristics indicate it may serve as a centrally active ligand of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), whose disruption of activity with organophosphate compounds (OP) leads to uncontrolled excitation and potentially life-threatening symptoms. To evaluate olesoxime as a binding ligand and reactivator of human AChE and BChE, we conducted in vitro kinetic studies with the active metabolite of insecticide parathion, paraoxon, and the warfare nerve agents sarin, cyclosarin, tabun, and VX. Our results showed that both enzymes possessed a binding affinity for olesoxime in the mid-micromolar range, higher than the antidotes in use (i.e., 2-PAM, HI-6, etc.). While olesoxime showed a weak ability to reactivate AChE, cyclosarin-inhibited BChE was reactivated with an overall reactivation rate constant comparable to that of standard oxime HI-6. Moreover, in combination with the oxime 2-PAM, the reactivation maximum increased by 10–30% for cyclosarin- and sarin-inhibited BChE. Molecular modeling revealed productive interactions between olesoxime and BChE, highlighting olesoxime as a potentially BChE-targeted therapy. Moreover, it might be added to OP poisoning treatment to increase the efficacy of BChE reactivation, and its cholesterol scaffold could provide a basis for the development of novel oxime antidotes.