Abstract Dielectrophoresis, the induced motion of dielectric particles in non-uniform electric fields, enables the separation of suspended bio-particles based on their dimensions or dielectric properties. This work presents a microfluidic system, which utilises a combination of dielectrophoretic (DEP) and hydrodynamic drag forces to separate Lactobacillus bacteria from a background of yeasts. The performance of the system is demonstrated at two operating frequencies of 10 MHz and 100 kHz. At 10 MHz, we are able to trap the yeasts and bacteria at different locations of the microelectrodes as they experience different magnitudes of DEP force. Alternatively, at 100 kHz we are able to trap the bacteria along the microelectrodes, while repelling the yeasts from the microelectrodes and washing them away by the drag force. These separation mechanisms might be applicable to automated lab-on-a-chip systems for the rapid and label-free separation of target bio-particles.