It has been found that lactic acid and hydrogen peroxide (H2O2) displayed co-operatively enhanced killing activity to pathogens. The synergistic effect was investigated with using several microbe species, suggesting that low concentration of lactic acid and H2O2 could kill both Gram-negative and Gram-positive bacteria or even fungal pathogens. To explore the mechanism of synergistic sterilization of lactic acid and H2O2, Escherichia coli DH5 alpha was used as the indicator bacteria. Lactic acid and H2O2 could generate hydroxyl radicals depending on the intracellular iron ions. The genomic DNA of treated cells was fractured and dispersed, and the orecA strain was more susceptive to the treatment, indicating that DNA damage was a cause of cell death. Furthermore, serious leakage of cell contents occurred in the treated cell, suggesting that the treatment also resulted in cell membrane permeability changes. This research shows that lactic acid-H2O2 consortium is a hopeful safety bactericide in agriculture or food production processes and provides a greater understanding of the mechanism of synergistic sterilization of lactic acid-H2O2 consortium in vivo.