The antibacterial activities of tungsten disulfide (WS2) nanosheets against two representative bacterial strains: Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) were evaluated by colony-forming unit (CFU) studies. The WS2 samples demonstrate a time and concentration dependent antibacterial activity (retardation of bacterial growth) for both bacterial strains. Morphology analyses reveal that WS2 nanosheets adhere to the bacterial surfaces, resulting in robust inhibition of cell proliferation once a bacterium is fully covered with this nanomaterial. More importantly, the intimate contact of WS2 nanosheets with a bacterium cell membrane can cause serious damage to the membrane integrity, and subsequently the cell death. On the other hand, the reactive oxygen species (ROS) generated by WS2 nanosheets are found to be modest regardless of the WS2 concentration, which is contradictory to the case of its structural analogue, MoS2, where ROS also play a significant role in its antibacterial activity. Taken together, our findings provide a detailed understanding of the antibacterial mechanism of WS2 nanosheets, which might help promote their potential applications in biomedical fields.