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Healthcare-associated infections have increasingly become problematic in the endoscopic procedures resulting in several severe diseases such as carbapenem-resistant Enterobacteriaceae (CRE)-related infections, pneumonia, and bacteremia. Especially, some bacterial strains are resistant to traditional antimicrobials. Therefore, the necessity of developing new antibiotics or management to deal with bacterial infections has been increasing. The current study combined a low concentration of glutaraldehyde (GTA) with near-infrared (NIR) light and 405-nm laser to entail antibacterial activity on Staphylococcus aureus biofilm. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and colony forming unit (CFU) counting were used to quantify the viable cells while fluorescent and scanning electron microscopic images were used to qualitatively evaluate the cell membrane integrity and structural deformation, respectively. Practically, S. aureus biofilm was highly susceptible (7% cell viability and 6.8-log CFU/cm² bacterial reduction for MTT assay and CFU analysis, respectively) to the combination of GTA (0.1%), NIR light (270 J/cm²), and 405-nm laser (288 J/cm²) exposure. GTA could form either DNA-protein or protein-protein crosslinks to inhibit DNA and protein synthesis. The NIR light induced the thermal damage on protein/enzymes while 405-nm laser could induce reactive oxygen species (ROS) to damage the bacterial membrane. Thus, the proposed technique may be a feasible modality for endoscope cleaning to prevent any secondary infection in the healthcare industry.