The aggregation and/or adhesion of bacterial cells is a serious disadvantage of electrophoretic separations. In this study, physicochemical surface characteristics of bacteria were measured to establish their role in bacterial adhesion and aggregation on the basis of electrophoretic behavior of different clinical strains of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria. The number and the shape of peaks obtained on the electropherograms were connected with the zeta potential measurements and in-line microscope observation using specially designed CE fluorescence stereomicroscope setup. These results suggest that the lower the zeta potential, the higher the number of smaller peaks detected. The direct microscopic observation of electrophoretic movement proved the presence of many small aggregates originating from individual or clustered bacterial cells. On the other hand, lower zeta potential was also observed for dead bacterial cells, which suggested that some of the peaks can be attributed to viable cells while the other to the dead ones.