The applicability of electric field as a pretreatment technique for inoculum preparation was confirmed in a previous study. In the present work, newly adopted electric pretreatment conditions were statistically optimized for the preparation of inoculum from anaerobic mixed cultures in dark fermentative H2 production via response surface methodology with a Box–Behnken design. Pretreatment conditions of applied voltage, distance of electrode, and reaction time were chosen as independent variables, while H2 yield was chosen as the response variable. Overall performance revealed that applied voltage and reaction time were slightly interdependent or significantly interactive influence on H2 yield, and they were more influential compared with electrode distance on H2 yield. In the confirmation test, H2 yield of 1.42 mol H2 per mol hexoseadded was recorded, corresponding with 94.7% of the predicted response value, under applied voltage of 11 V, distance of electrode of 5 cm, and reaction time of 23 min. Although the H2 yield under electric pretreatment was similar with the heat pretreated yield, the energy consumption efficiency was about 40‐fold lower. Therefore, it may be concluded that electric pretreatment is a potent alternate technique for inoculum preparation method in terms of energy consumption and easy applicability in the field with high effectiveness. Copyright © 2014 John Wiley & Sons, Ltd.