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In this paper, we report experimental results aimed at the quantitative description of bacterial inactivation using a low-energy (<100 kV) pulsed-electron beam. The initial step was to demonstrate the feasibility of a secondary emission electron gun (SEEG) on the area of sterilization/decontamination, which is quantitatively related to the survival characteristics of bacteria. The survival characteristic of most common type of bacterium Escherichia coli JM 109 (E. coli) was studied in an atmospheric pressure decontamination chamber under the increased gun voltage as well as varied pulsed-electron-beam parameters such as current density, pulse width, and repetition rate. A complete inactivation of E. coli was achieved by a single-electron-beam pulse at an accelerating gun voltage of 85 kV in time duration of 5 μs or by five electron beam pulses of the same time duration at a voltage of 77 kV. Several inherent advantages including an efficient bacterial inactivation have been provided as a basis for utilization of SEEG (or low-energy electron beam in general) as a decontamination tool in various biological and medical applications.