A new triglyceride-free propofol microemulsion for intravenous injection was formulated using nonionic surfactants, poloxamers and polyethylene glycol 660 hydroxystearate. The aim of this investigation was to evaluate the formulation for storage stability, antimicrobial activity, toxicity and preclinical efficacy. The results were compared to the characteristics obtained for the most commonly used formulation of propofol (Diprivan$^{(R)}$). The mean particle diameter of the microemulsion was less than 100 nm so that it could be readily sterilized using a 0.22 ${mu}M$ membrane at room temperature. The microemulsion formulation demonstrated enhanced stability compared to the marketed macroemulsion formulation. In a stress storage condition, it was physicochemically stable for at least 40 months. This new formulation showed higher antimicrobial activity, lower risk of hyperlipidemia and better tolerability than Diprivan$^{(R)}$). In preclinical studies, the efficacy and pharmacokinetic profile of the microemulsion were similar to those of Diprivan$^{(R)}$). Nevertheless, the administration of the microemulsion caused considerably low histamine release compared to the macroemulsion. Based on these results, the newly developed microemulsion of propofol appeared to have several advantages and, thus, could be an alternative to the fat macroemulsions of propofol.