Abstract: The elimination of biofilms is a crucial step in controlling hospital-acquired infections. Once biofilms colonize luminal instruments, it is difficult to remove them using traditional disinfection methods. Conventional disinfection approaches now face a series of challenges, including microbial resistance, corrosiveness, cytotoxicity, residual disinfection byproducts, and environmental pollution. Therefore, developing novel disinfection technologies specifically targeting biofilm removal is vitally important. New disinfection technologies, such as slightly acidic electrolyzed water, plasma technology, surface modification techniques, nanomaterial-based disinfection, bacteriophage disinfection, and enzymatic disinfection, are constantly emerging. These technologies exhibit excellent performance against biofilms by leveraging the synergistic effects of multiple mechanisms, including the reactive oxygen species (ROS) burst, photocatalytic oxidation, physical disruption, and biological targeting. This review summarizes the characteristics, underlying mechanisms, and potential application scenarios of these novel disinfection technologies, with a particular focus on their effects against biofilms formed by common pathogenic bacteria on surfaces in hospital settings. It aims to provide a reference basis for the practical application and translation of these disinfection technologies and the development of new disinfection strategies.