The major challenge in bacterial infection in clinical settings is the development of antimicrobial materials in the treatment of drug-resistant bacteria. Herein, we report a new strategy for efficient near-infrared radiation (NIR) photothermal sterilization and focal infection treatment by acetylcysteine-modified Prussian blue nanoparticles (AC-PB). Specifically, AC-PB is fabricated as a multifunctional therapeutic agent via a co-precipitation approach, where PB acts as an effective photothermal agent and AC could prevent the formation of bacteria cluster in biofilms and the bacterial adhesion on tissues to reduce the secretion of mucus and improve the efficacy. AC-PB shows strong synergistic photothermal sterilization ability in a concentration-dependent manner by using 980 nm NIR laser. 50 μg/mL of AC-PB can eliminate up to 74% of Gram-positive Staphylococcus aureus and up to 75% of Gram-negative Escherichia coli, while irradiation of 980 nm is minimally cytotoxic to mammalian cells. The NIR radiation can be efficiently converted into local heat by subcutaneous injection of AC-PB to kill bacteria effectively in vivo to treat a focal infection. The antibacterial mechanism suggests that AC can destroy bacteria-based biofilms, while the photothermal effect driven by NIR may break the lipids on cellular membrane. Thus, this work may provide a promising strategy for highly effective eradication of bacteria in clinics.