The assessment of capillary electrophoresis for separation and identification of microorganisms is described in this article. The work brings a comparison of the application of uncoated capillaries modified with poly(ethylene oxide) and coated capillaries for the separation of model microorganisms Saccharomyces cerevisiae and Escherichia coli with Tris-borate-EDTA and Tris-citrate-fructose as electrolytes. The best separation was achieved in the coated capillary using 1 mM Tris-citrate-fructose buffer, pH 6.9. A simple identification based on the migration time and UV spectrum was found unsatisfactory and thus a method based on a post-separation cultivation and sequential analysis was developed. Off-line combination with mass spectrometric analysis with the use of desorption electrospray was shown to be an interesting alternative in a study of microorganisms. Mass spectra allowed distinguishing among analyzed cells.