This study aimed to investigate the antibacterial properties and mechanisms of a high-voltage static electric field (HVEF) in Acinetobacter johnsonii, which were assessed from the perspective of biochemical properties and stress-related genes. The time/voltage-kill assays and growth curves showed that an HVEF decreased the number of bacteria and OD600 values. In addition, HVEF treatment caused the leakage of cell contents (nucleic acids and proteins), increased the electrical conductivity and amounts of reactive oxygen substances (ROS) (16.88 fold), and decreased the activity of Na+ K+-ATPase in A. johnsonii. Moreover, the changes in the expression levels of genes involved in oxidative stress and DNA damage in the treated A. johnsonii cells suggested that HVEF treatment could induce oxidative stress and DNA sub-damage. This study will provide useful information for the development and application of an HVEF in food safety.