Room-temperature ionic liquids (ILs) can be used as reaction media for nonaqueous biocatalysis. However, the purity of ILs should be considered to understand the influence of ILs on enzyme activity. The major impurities in ILs are water and halide. In the transesterification of benzyl alcohol with vinyl acetate, the optimal water activities for lipases in [Omim][Tf2N] were similar to those in organic solvents. The chloride impurity in [Omim][Tf2N] seriously influenced the activity of lipase. In this work, the effect of ILs on lipase activity was investigated under controlled initial water activity and low halide content. The activity of lipase was highly dependent upon the anion structure of ILs. The initial reaction rate of lipases followed the order [Tf2N]- >[PF6]- >[TfO]- >[SbF6]- ≒[BF4]-. All tested lipases showed the highest activities in ILs containing [Tf2N] anion. Particularly, [AAIM][Tf2N] was shown as a suitable reaction medium for biocatalysis. Lipozyme IM showed the highest activity in this IL among tested ILs. Thermal stability of lipase was also investigated. The higher thermal stability of Novozym 435 was obtained in hydrophobic and water-immiscible ILs such as [Bmim][Tf2N], [Edmim][Tf2N], and [Bmim][PF6].