The high-voltage electrostatic field (HVEF) has been proposed as an efficient and convenient strategy for microbial inactivation, playing a crucial role in ensuring urban safety and people's lives and health. However, the effects of the underlying antibacterial molecular mechanism on specific functional capabilities are largely unknown. Here, we systematically investigated the molecular mechanism underlying the inactivation effect of an HVEF against E. coli with a wire-plate-type device. Our experimental analysis revealed that the antibacterial effects primarily stemmed from the local alteration of cell membrane integrity and permeability, which further induced a series of oxidative damage events, including decreased SOD activity, increased ROS levels and MDA content, and, eventually, apoptosis. Theoretically, this process is mediated mainly by energy metabolism, cell motility and membrane transport signaling, as suggested by a multiomic analysis. Through quantitative methods, we showed that FliC, a key flagellar protein, plays a very important role in this process and that the quantity of fliC present on cells influences the HVEF tolerance. These results together reveal the previously unknown mechanism underlying the antibacterial effect of HVEFs and suggest that fliC activity and cell motility are novel components of this mechanism that distinguish HVEF-resistant bacteria from normal bacteria.