Ionic Liquid (ILs) in oil microemulsions (IL/O MEs) have emerged as a potential carrier for transdermal drug delivery, particularly for sparingly soluble drug molecules, such as acyclovir and methotrexate. However, less attention was paid in the selection of non-toxic and biodegradable ILs. In this paper, we report new IL/O MEs with ammonium acetate ILs, which can dissolve acyclovir and methotrexate significantly. Microemulsions were composed of a mixture of nonionic surfactants, polyoxyethylene sorbitan monooleate (Tween-80), and sorbitan laurate (Span-20)/ILs/oil isopropyl myristate (IPM). Three Its called 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), triethylammonium acetate ([TEA][OAc]) and diethylammonium acetate ([DEA][OAc]) were selected due to their non-toxic property. Based on the pseudo-ternary diagrams constructed, it was evident that Span-20 played the key role as a surfactant while Tween-80 acted as the co-surfactant, showing optimal dissolution of the ILs into the system prepared with a ratio of 1:3 (Tween 80:Span20). This binary mixture also depicted the lowest viscosity, which is favorable as a carrier agent in drug delivery. The incorporation of various ILs into microemulsions resulted in a particle size ranging from 11 to 39.7 nm at diverse drug loading capacity. The formulation with IL [TEA][OAc] and IL [DEA][OAc] displayed substantial drug carrying capacity and stability compared to the commercially available IL (EMIM)[OAc] loaded system.