Herein, we report a facile approach for the preparation of two-dimensional iodine nanosheets (2D iodine NSs) with good stability and high biocompatibility via an aqueous solvent-assisted ultrasonic route. Due to the large specific surface area of the 2D morphology, iodine NSs effectively interact with bacterial membranes and destroy bacterial integrity, as well as further damaging intracellular DNA, showing prominent antibacterial activity against S. aureus in vitro and in vivo.