We have fabricated a microfluidic device based on a novel geometrical arrangement of lateral metal electrodes and a patterned insulator. Our device combines the concept of insulator-based “electrodeless” dielectrophoresis with multiple frequencies to achieve focusing and continuous separation of dielectric particles flowing through a channel. The opposition of two DEP- force fields defines a position of equilibrium for the dielectric particles placed in these fields. The equilibrium is externally controllable by the applied signals at different frequencies and is a function of the dielectric properties of the particles and their surrounding medium. Opposite DEP-forces were used to accurately focus a stream of beads and yeast cells at different positions across the channel by simply adjusting potentials and frequencies. The same opposition of forces was then extended to the continuous separation of particles with different dielectric properties. A numerical calculation allowed extracting dielectric properties of the particles from their equilibrium positions.