Nano-titania is widely used in the food industry due to its efficient antimicrobial activity. However, the mechanism of microbial toxicity of nano-titania is poorly known. Here, nano-TiO2 has been fabricated by microwave-irradiation chemistry, a new method, and then tested for antimicrobial activity. Mutagenicity of nano-TiO2 was evaluated using Salmonella typhimurium histidine-auxotrophic strains. The reactive oxygen generation test was performed using 2,7-dichlorofluorescein diacetate dye. To test membrane permeabilization, E. coli cultures were grown in nutrient broth at an optical density of 0.3–0.5 at 610 nm, harvested by centrifugation at 11,000g for 10 min, washed and resuspended in 0.5 % NaCl solution. We also analyzed superoxide formation and membrane integrity, and we used scanning electron microscopy. Results show that nano-TiO2 has a minimum inhibitory concentration of 15 µg/mL, and a minimum bactericidal concentration of 20 µg/mL for E. coli. The bacterial inner wall was ruptured, and cytoplasmic content was released after 5 min of treatment in a dose-dependent manner. Notably, superoxide formation was not observed, which establishes the fact that reactive oxygen generation and alteration of membrane integrity, as well as permeability, is the major mechanism of antimicrobial activity of nano-TiO2. © 2016, Springer International Publishing Switzerland.