A-series agent A-234 is a new generation nerve agent that was included in the Chemical Weapons Convention following the poisoning of a Russian spy and his daughter in 2018. This study characterized A-234's stability, inhibitory potential towards human cholinesterases (AChE and BChE), reactivation ability, acute toxicity, and therapeutic approaches. A-234 was found to be a potent inhibitor of both AChE and BChE, with IC50 values of 0.101 μM and 0.036 μM, respectively. However, none of the five marketed oximes showed significant reactivation ability towards A-234-inhibited cholinesterases. The acute toxicity of A-234 is comparable to that of VX, and atropine and diazepam effectively mitigate its lethality. Molecular dynamics simulations suggest that the oximes are weak nucleophiles, which may explain their inability to reactivate A-234-inhibited cholinesterases. The study provides essential experimental preclinical data on A-234.A-series agent A-234 is a new generation nerve agent that was included in the Chemical Weapons Convention following the poisoning of a Russian spy and his daughter in 2018. This study characterized A-234's stability, inhibitory potential towards human cholinesterases (AChE and BChE), reactivation ability, acute toxicity, and therapeutic approaches. A-234 was found to be a potent inhibitor of both AChE and BChE, with IC50 values of 0.101 μM and 0.036 μM, respectively. However, none of the five marketed oximes showed significant reactivation ability towards A-234-inhibited cholinesterases. The acute toxicity of A-234 is comparable to that of VX, and atropine and diazepam effectively mitigate its lethality. Molecular dynamics simulations suggest that the oximes are weak nucleophiles, which may explain their inability to reactivate A-234-inhibited cholinesterases. The study provides essential experimental preclinical data on A-234.