Dehydrogenated steroids are usually more effective in treating diseases, compared to their precursors. In this study, toxicity of six ionic liquids to the Arthrobacter simplex was investigated to evaluate the possibility of dehydrogenation of 11α-hydroxy-16,17α-epoxy progesterone in ionic liquid / water biphasic system. Results indicated that ionic liquids displayed higher toxicities to the A. simplex. The followed bio-dehydrogenation in biphasic system, as well as the immobilization investigations in the corresponding system, showed the lower conversion ratio of 11α-hydroxy-16,17α-epoxy progesterone. This maybe indicated the unsuitability of the imidazole-based ionic liquid for the dehydrogenation of A. simplex in the biphasic system.