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Recently, silver (Ag) and copper (Cu) have been incorporated into a titanium (Ti) surface to realize their antibacterial property. This study investigated both the durability of the antibacterial effect and the surface change of the Ag- and Cu-incorporated porous titanium dioxide (TiO₂) layer. Ag- and Cu-incorporated TiO₂ layers were formed by micro-arc oxidation (MAO) treatment using the electrolyte with Ag and Cu ions. Ag- and Cu-incorporated specimens were incubated in saline during a period of 0–28 days. The changes in both the concentrations and chemical states of the Ag and Cu were characterized using X-ray photoelectron spectroscopy (XPS). The durability of the antibacterial effects against <i>Escherichia coli</i> (<i>E. coli)</i> were evaluated by the international organization for standardization (ISO) method. As a result, the Ag- and Cu-incorporated porous TiO₂ layers were formed on a Ti surface by MAO. The chemical state of Ag changed from Ag₂O to metallic Ag, whilst that of Cu did not change by incubation in saline for up to 28 days. Cu existed as a stable Cu₂O compound in the TiO₂ layer during the 28 days of incubation in saline. The concentrations of Ag and Cu were dramatically decreased by incubation for up to 7 days, and remained a slight amount until 28 days. The antibacterial effect of Ag-incorporated specimens diminished, and that of Cu was maintained even after incubation in saline. Our study suggests the importance of the time-transient effects of Ag and Cu on develop their antibacterial effects.