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Effective photosensitizers (PSs) are at the heart of the key role of photo-driven antibacterial therapy. However, relative high temperature required for bactericidal activity, stimulating at multiple wavelengths, and multi-step synthesis of the photothermal and photodynamic agents challenge their applications in antibacterial treatment. In this study, silver nanoparticles (AgNPs) functionalized by kaempferol (Kae) are implanted into Prussian blue (PB) to construct photo-responsive nanocubes (AgPB) by a simple synthetic process. The as-synthesized AgPB nanocomposites were found to have an average size of ∼140 nm, and the particle size range of the loaded-AgNPs was 5–15 nm. The structure of AgPB was characterized, and the photothermal and photodynamic evaluation of antibacterial activity of AgPB was investigated under single 808 nm near-infrared (NIR) light. By adjusting the doping ratio of AgNPs, the band gap can be tuned from 2.78 to 2.56 eV to further enhance the photothermal conversion of AgPB and its ability to generate reactive oxygen species (ROS). When compared with AgPB without NIR illumination, in vitro antibacterial studies by using Escherichia coli and Staphylococcus aureus showed that AgPB under 808 nm NIR light could rapidly heat up to 50 °C, enhance the formation of ROS, and promote the ion release amount of Fe2+, Fe3+, and Ag+ to increase oxidative stress, leading to inhibition of bacterial proliferation significantly. Both E. coli and S. aureus were completely killed within 10 min. Moreover, the biofilm formation was remarkably inhibited, and the eradication ratio against E. coli and S. aureus biofilms was 69.73 and 60.01%, respectively. In addition, the hemolytic activity test proved that AgPB had good biocompatibility. The results showed that AgPB with triple bactericidal modalities could expand the application of PSs and serve as a promising nanocomposite for antibacterial therapy. [ABSTRACT FROM AUTHOR]