Au@Ag nanoparticles (NPs) were recently found to display giant two-photon photoluminescence (2PPL) enhancement, with an enhancement factor up to 815 fold upon aggregate formation. Based on this finding, two-photon imaging of bacteria by Au@Ag NPs under near-infrared (NIR) femtosecond laser pulses was demonstrated in this study, as positively charged Au@Ag NPs can form aggregates on the negatively charged bacterial surface, yielding strong 2PPL emission. The aggregation-enhanced 2PPL of Au@Ag NPs stemmed from higher two-photon excitation efficiency, implying strong two-photon photothermal effects. Au@Ag NPs showed strong antibacterial activity (minimum inhibition concentration as low as 7.5 pM against Staphylococcus aureus) and negligible toxicity to human dermal fibroblasts. Their bactericidal activity was further enhanced under NIR irradiation due to strong two-photon photothermal effects. Au@Ag NPs effectively removed 85% of the notorious bacterial biofilm within 4 min under NIR irradiation. These Au@Ag NPs can potentially be used as imaging and antibacterial agents.