A repeatable preconcentration electrophoretic methodology for the analysis of bacteria was developed. This method is based on an isotachophoretic mode coupled with a simultaneous hydrodynamic-electrokinetic injection in conditions of field-amplified sample injection. This electrophoretic method allows the quantification of Enterobacter cloacae (studied as a model of Gram negative bacteria) with a limit of detection of 2 × 104 cells/mL. With the optimized conditions, a preconcentration factor of about 500-fold was obtained as compared to a standard hydrodynamic injection. The RSD (n = 5) on the migration time and on the peak area were 3% and 5%, respectively. This capillary electrophoretic methodology has been applied for the quantification of microbes in natural water (river and natural spring waters). Filtration of the sample prior to injection was required to remove ions present in the water and to keep the field-amplified sample injection condition at the injection. Filtrated bacteria were then recovered in terminating electrolyte diluted 10 times with water. Good agreements were obtained between cellular ATP measurements and the proposed CE methodology for the quantification of bacteria in waters.