To combat multidrug-resistant bacteria, researchers have poured into the development and design of antimicrobial agents. Here, low-cost two-dimensional (2D) antibacterial material titanium monoxide nanosheets (TiO NSs) were prepared by an ultrasonic-assisted liquid-phase exfoliation method. When cultured with bacteria, TiO NSs showed intrinsic antimicrobial capacity, possibly due to membrane damage caused by the sharp edges of TiO NSs. Under near-infrared (NIR) laser irradiation, TiO NSs showed high photothermal conversion efficiency (PTCE) and sterilization efficiency. By combining these two antibacterial mechanisms, TiO NSs exhibited a strong killing effect on Gram-negative Escherichia coli (E. coli) and Gram-positive methicillin-resistant Staphylococcus aureus (MRSA). Especially after treatment with TiO NSs (150 μg mL-1) +near-infrared (NIR) light irradiation, both bacteria were completely killed. In vivo experiments on wound repair of bacterial infection further confirmed its antibacterial effect. In addition, TiO NSs had no obvious toxicity or side effects, so as a kind of broad-spectrum 2D antibacterial nanoagent, TiO NSs have broad application prospects in the field of pathogen infection.