Ionic liquids are compounds that composed only of ions and are liquid at room temperature. Thus, it is normally named room temperature ionic liquid (RTIL). In this study, the application of RTILs to the extractive fermentation of biomaterials was investigated as a substitute of organic solvents. The relative toxicity of the RTILs on the growth of E. coli was tested. The inhibition of cell growth in the presence of various ionic liquids was measured using solid and liquid culture, and EC50 of each RTILs was calculated. The number of viable and total cells was measured by the number of colonies and optical density, respectively. Effective concentrations of toxicity (EC50) in these tested systems were similar with conventional solvents, such as acetone, acetonitrile, and ethanol. The viability of E. coli was affected by the polarity and ionic properties of ionic liquids. The resistance of the microorganisms against ionic liquids was different with the cations and anions composing ionic liquids. No general influence of the anionic compound of the ionic liquids was found on toxicity comparing with distinctive influence of cationic moiety.