This study investigates the effects of pulsed electric fields on the inactivation of trophozoite form of Naegleria lovaniensis Ar9M-1 in batch and flow processes, systematically examining the lethal effect of field strength, pulse duration, number of pulses, and pulse frequency. Our results show that amoebae eradication is modulated by pulse parameters, composition of the pulsing medium, and physiological state of the cells. Cell survival is not related to the energy delivered to the cell suspension during the electrical treatment. For a given energy a strong field applied for a short cumulative pulse duration affects viability more than a weak field with a long cumulative pulsation. We also determine the optimal electrical conditions to obtain an inactivation rate higher than 95% while using the least energy. Flow processes allow to treat large-scale volumes. Our results show that the most efficient flow process for amoeba eradication requires a field parallel to the flow. Pulsed electric fields are a new and attractive method for inactivating amoebae in large volumes of fresh water.