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Dielectrophoresis (DEP) can be an effective tool to show the physiological change of bacterial cells. The behavior of bacterial cells under an electric field is complicated due to the combined effects of electrokinetic phenomena. This paper presents the study of the electrokinetic behavior of heat-treated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) cells for a cell counting method. Through numerical and experimental study, heat-treated BCG cells are compared with control BCG cells. At various frequencies with the medium conductivity of 0.07 S/m, the equilibrium positions of both control- and heat-treated cells are analyzed in the combined fields of DEP and AC electroosmosis (ACEO). As DEP changes from negative to positive in electroosmotic flow, the equilibrium position of cells is bifurcated from the upper center between two electrodes onto the edges of both electrodes. It was found that the cells floating on electrodes should not be counted as attracted cells because the floating was resulted from the combined effect of the negative DEP and ACEO. According to the analysis, an optimum frequency is proposed to differentiate control cells from heat-treated cells using a cell counting method. The presented study will offer physical insight for the cell counting to differentiate live and dead Mycobacterium bovis BCG cells treated with heat and drugs.