Nanostructured magnesium oxide (MgO) is a new kind of environment‐friendly antimicrobial agents with high efficiency, non‐toxicity and relatively low cost. Herein, MgO nanoplates were synthesized by a simple hydrothermal‐calcination method. Antibacterial test showed that the MgO nanoplates calcinated at 450 °C performed the best antibacterial activity, with the antibacterial ratio higher than 99.9 % against Escherichia coli. Based on the results of structural characterization and superoxide radical (.O2−) detection, the excellent antibacterial activity of MgO was attributed to the high content of oxygen vacancies, which could result in more .O2− production. In addition, the MgO nanoplates exhibited good antibacterial performance at low concentration, and also performed long‐term antibacterial activity. Our method provides a strategy for regulation of surface defects and improvement of antibacterial activity on the pure MgO nanoplates.