Magnesium oxide nanoparticles (MgO NPs) were obtained by the calcination of precursor microparticles (PM) synthesized by a novel triethylamine-based precipitation method. Scanning electron microscopy (SEM) revealed a mean size of 120 nm for the MgO NPs. The results of the characterizations for MgO NPs support the suggestion that our material has the capacity to attack, and have an antibacterial effect against, Gram-negative and Gram-positive bacteria strains. The ability of the MgO NPs to produce reactive oxygen species (ROS), such as superoxide anion radicals (O2•-) or hydrogen peroxide (H2O2), was demonstrated by the corresponding quantitative assays. The MgO antibacterial activity was evaluated against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria, with minimum inhibitory concentrations (MICs) of 250 and 500 ppm on the microdilution assays, respectively. Structural changes in the bacteria, such as membrane collapse; surface changes, such as vesicular formation; and changes in the longitudinal and horizontal sizes, as well as the circumference, were observed using atomic force microscopy (AFM). The lipidic peroxidation of the bacterial membranes was quantified, and finally, a bactericidal mechanism for the MgO NPs was also proposed.