Antibacterial agents are an important tool in the prevention of bacterial infections. Inorganic materials are attractive due to their high stability under a variety of conditions compared to organic antibacterial agents. Herein tungsten oxide nanodots (WO3-x), synthesized by a simple one-pot synthetic approach, were found to exhibit strong antibacterial capabilities. The analyses with colony-forming units (CFU) showed an excellent antibacterial activity of WO3-x against both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) strains. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed clear damages to the bacterial cell membranes, which was further confirmed by molecular dynamics simulations. Additionally, exposure to simulated sunlight was found to further increase the germicidal activity of WO3-x nanodots, a 30 min exposure to sunlight combined with 50 μg/mL WO3-x nanodots showed a 70% decrease in E. coli viability compared to without exposure. Electron spin resonance spectroscopy (ESR) was used to elucidate the underlying mechanism of this photocatalytic activity through the generation of hydroxyl radical species. The cell counting kit-8 (CCK-8) and the live/dead assay were further employed to evaluate the cytotoxicity of WO3-x nanodots on eukaryotic cells, which demonstrated their general biocompatibility. In summary, our results suggest WO3-x nanodots have considerable potential in antibacterial applications, while also being biocompatible at large.