The widespread use of antibiotics has led to the emergence of multidrugresistant bacterial strains, and therefore a current concern for food safety and human health. The interest for new antimicrobial substances has been focused toward metal oxide nanoparticles. Specifically, titanium dioxide (TiO 2) has been considered as an attractive antimicrobial compound due to its photocatalytic nature and because it is a chemically stable, non-toxic, inexpensive, and Generally Recognized as Safe (GRAS) substance. Several studies have revealed this metal oxide demonstrates excellent antifungal and antibacterial properties against a broad range of both Gram-positive and Gram-negative bacteria. These properties were significantly improved by titanium dioxide nanoparticles (TiO 2 NPs) synthesis. In this chapter, latest developments on routes of synthesis of TiO 2 NPs and antimicrobial activity of these nanostructures are presented. Furthermore, TiO 2 NPs favor the inactivation of microorganisms due to their strong oxidizing power by free radical generation, such as hydroxyl and superoxide anion radicals, showing reductions growth against several microorganisms, such as Escherichia coli and Staphylococcus aureus. Understanding the main mechanisms of antimicrobial action of these nanoparticles was the second main purpose of this chapter.