Effective and safe application of antibacterials has always been an important aspect for their usage. High-efficiency photocatalytic technology driven by visible light for antibacterial action constitutes a practical solution for antibacterial agents and will not harm the human body or the environment. While most studies on β-Bi2O3 materials with good photocatalytic properties under visible light are conducted in the field of optoelectronics, their potential and mechanism as photocatalytic antibacterial agents have not yet been fully explored. Herein, we report the performance of sheet-like metastable β-Bi2O3 material with rich oxygen vacancies and high electron-hole separation efficiency in antibacterial processes, as well as a preliminary exploration of its antibacterial mechanism. The results revealed that the antibacterial activity of the product against E. coli greatly improved in comparison with commercially available α-Bi2O3 owing to its excellent structure and optical properties. In addition, gradient experiments and scavenger experiments have confirmed that the main antibacterial effect of β-Bi2O3 originates from reactive oxygen species (ROS), and the superoxide radical, ·O2−, of generated ROS is the key reactive species in the antibacterial process. Through the detection of lipid peroxidation and bacterial respiratory-chain dehydrogenase activity, several pathways were identified for the excellent antibacterial activity of the product.