The vast majority of prokaryotic species are difficult or impossible to culture in laboratories, which makes it difficult to study these organisms using conventional biochemical techniques. Methods that enable the physical isolation of single prokaryotic cells would thus facilitate the characterization of previously unstudied organisms by eliminating or reducing the need for cultivation. Most current methods for single-cell isolation were designed for eukaryotic cells mainly of mammals, which are non-motile and much larger than prokaryotic cells. We therefore developed a micro-chamber array–based method for the isolation of single prokaryotic cells using dielectrophoresis. Here, we demonstrated the applicability of the method using two prokaryotic species, Escherichia coli (Bacteria) and Haloferax volcanii (Archaea), which differ both in size and biochemical composition. Our results showed that cells of either organism are trapped with an applied electric field of 5 to 20 MV/m and 50 kHz to 3 MHz, and that the optimum combination of dielectrophoresis voltage and frequency depends on the cell type. We suggest that this technique is useful for trapping single cells of diverse prokaryotic species.