The inactivation of Escherichia coli using moderate electric fields (MEF) below 25 °C, was investigated. Keeping the temperature always below 25 °C demonstrated that electric fields are involved in the inactivation of E. coli, without possible synergistic temperature effects. Electric fields above 220 V cm−1 promoted death rates of 3 log10 cycles of E. coli in less than 6 min, and even higher rates at greater electric fields, while presumably overcoming the thermal degradation caused by conventional high temperature treatments. A non-thermal model was proposed that successfully describes the E. coli death kinetics under this treatment. SEM observations of E. coli cells after the exposure to the MEF treatment, revealed changes at the cell membrane level, indicating a possible cause for the cell death rates. These results show that this treatment holds potential for sterilization of thermolabile products (e.g. serum and other physiological fluids, food products), by itself or as a complement of the traditional heat-dependent techniques.