A facile one-pot sonochemical synthesis of Cu-, Ag-, and hybrid Cu/Ag-based polydopamine nanoparticles (Cu-, Ag-, and Cu/Ag-PDA-NPs) and the mechanisms by which they exert antibacterial and antibiofilm activities are reported. We showed that the nanoparticles are spherical with a core shell structure. Whereas Cu is chelated to the shell of Cu-PDA-NPs in oxidation states of +1/+2, the core of Ag-PDA-NPs is filled with elemental Ag degrees. Sonochemical irradiation of dopamine in the presence of both Cu2+ and Ag+ generates hybrid Cu/Ag-PDA-NPs, whose shells are composed of Cu-chelated PDA with Ag degrees in the core. The redox potential of the metals was found to be the main determinant of the location and oxidation state of the metals. Leaching studies under physiological conditions reveal a relatively fast release of Cu ions from the shell, whereas Ag leaches very slowly from the core. The metal-containing PDA-NPs are highly microbicidal and exhibit potent antibiofilm activity. The combination of both metals in Cu/Ag-PDA-NPs is especially effective against bacteria and robust biofilms, owing to the dual bactericidal mechanisms of the metals. Most importantly, both Ag- and Cu/Ag-PDA-NPs proved to be significantly more antibacterial than commercial Ag-NPs while exhibiting lower toxicity toward NIH 3T3 mouse embryonic fibroblasts. Mechanistically, the metal containing PDA-NPs generate stable PDA-semiquinone and reactive oxygen species under physiological conditions, which contribute at least partly to the antimicrobial activity. We also demonstrated that simple treatment of surfaces with Ag-PDA-NPs converts them to antibacterial, the activity of which was preserved even after prolonged storage under ambient conditions.