Microscale electrokinetic techniques have great potential for the separation and sorting of microorganisms, and could solve the need for rapid and early detection of pathogens in medical diagnostics and food safety applications. Presented here is the application of micro particle image velocimetry for the characterization of electrokinetic transport of three types of microorganisms: Escherichia coli, Ankistrodesmus spiralis, and Saccharomyces cerevisiae. The electrokinetic behavior of these microorganisms was characterized employing a straight glass microchannel and direct current electric fields (50-300 V cm(-1)). The effects of the type and size of microorganism, electric field magnitude, and suspending medium characteristics were analyzed. Additionally, electrokinetic differentiation was achieved when a sample containing a mixture of the three types of microorganisms was analyzed by generating an electropherogram in one minute, identifying that three different species were present. These results demonstrate that fast and effective detection, and differentiation of intact microorganisms can be achieved employing microscale electrokinetic techniques.