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Insulator‐based dielectrophoresis (iDEP) was utilized to separate and concentrate selectively mixtures of two species of live bacteria simultaneously. Four species of bacteria were studied: the Gram‐negative Escherichia coli and the Gram‐positive Bacillus subtilis, B. cereus, and B. megaterium. Under an applied direct current (DC) electric field all the bacterial species exhibited negative dielectrophoretic behavior. The dielectrophoretic separations were carried out in a glass microchannel containing an array of insulating posts. The insulating posts in the microchannel produced nonuniformities in the electric field applied along the channel. Mixtures of two species of bacteria were introduced into the microchannel and the electric field was applied. The bacterial species exhibited different dielectrophoretic mobilities under the influence of the nonuniform field. From these experiments a trapping order was established …