Reversible cholinesterase inhibitors have been investigated to address prophylactic and therapeutic outcomes against organophosphorus nerve poisoning, and procyclidine is concomitantly used to compensate for their adverse effects. We designed double-layer patches with microneedles composed of a cream layer for immediate release and a hyaluronic acid-based thin solid layer for sustained release. Rivastigmine and procyclidine were added to the two layers with different doses, and their transdermal absorption was explored in guinea pigs for the first time. The contents of hyaluronic acid and the number of microneedles were also examined to determine the optimal patches, which would achieve the mean target plasma concentrations of the two drugs for 72 h. Plasma concentrations of rivastigmine and procyclidine were determined using a validated HPLC-MS/MS method, and acetylcholinesterase (AChE) activity was monitored and correlated with plasma rivastigmine concentrations. The systemic exposure of both drugs was successfully characterised in guinea pigs, and the change was relevant in terms of different patches with microneedles. The inhibitory effect on AChE was prolonged despite the decay of rivastigmine concentrations. Finally, the preparation of a double-layer patch with microneedles was suggested, and the present results would help explain the prophylactic and therapeutic effects of rivastigmine and procyclidine against biochemical weapons.