Abstract In our previous paper we have modelled a dielectrophoretic force (DEP) and cell particle behavior in a microfluidic channel (Weber MU et al 2023 Chip for dielectrophoretic microbial capture, separation and detection I: theoretical basis of electrode design Nanotechnology this issue). Here we test and confirm the results of our modeling work by experimentally validating the theoretical design constraints of the ring electrode architecture. We have compared and tested the geometry and particle capture and separation performance of the two separate electrode designs (the ring and dot electrode structures) by investigating bacterial motion in response to the applied electric field. We have quantitatively evaluated the electroosmosis (EO) to positive DEP (PDEP) transition in both electrode designs and explained the differences in capture efficiency of the ring and dot electrode systems. The ring structure shows 99% efficiency of bacterial capture both for PDEP and for EO. Moreover, the ring structure shows an over 200 faster bacterial response to the electric field. We have also established that the ring electrode architecture, with appropriate structure periodicity and spacing, results in efficient capture and separation of microbial cells. We have identified several critical design constraints that are required to achieve high efficiency bacterial capture. We have established that the spacing between consecutive DEP traps smaller than the length of the depletion zone will ensure that the DEP force dominates bacterial motion over motility and Brownian motion.