The effect of a pulsed electric field (PEF) on Staphylococcus epidermidis was investigated by using an atomic force microscopy (AFM) image and force measurement under liquid. The changes in the bacterial envelope were probed in situ before and after applying different dosages of PEF. Our results indicated that PEF induced the changes of bacterial peptidoglycan layer and the exposure of plasma membrane. This conclusion was further confirmed by two control experiments: the effect of the lysozyme or heat on the bacterial envelope. Furthermore, our results demonstrated that AFM analysis including image and force measurement is helpful to explain the relationship between the chemical composition change of the cellular envelope and the external stimulation.