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In recent years, infectious diseases, specifically those that are caused by pathogens, have seen a dramatic proliferation due to resistance to multiple antibiotics, opening the colony by opportunistic pathogens. Nanotechnology and tissue engineering have been applied in the development of new antimicrobial therapies, capable of fighting opportunistic infections. In the medical field, research on antimicrobial properties of metal oxide nanoparticles have emerged to find new antimicrobial agents as an alternative against resistant bacteria. The metal oxides, particularly those formed by transition metals are compounds with electronic properties, and most magnetic phenomena involve this type of oxides. Nanoparticles-based metal oxide properties such as shape, size, roughness, zeta potential and their large surface area, make oxides ideal candidates to interact with bacteria and able to have an antimicrobial effectiveness. The aim of this chapter is to offer an updated panorama about the relationships between the use of metal oxide nanoparticles in the medical field, with an emphasis on their role as antimicrobial agents and the properties that influence their antimicrobial response. In addition, the mechanism of nano-antimicrobial action is described and the importance of using in vitro test methods, adopted by leading international regulatory agencies, that can be used to determine the antimicrobial activity of the metal oxide nanoparticles.