Cystoscope cleaning and sterilization are one of the most important procedures to prevent infection in urological surgery. The current cleaning techniques have demonstrated lengthy process times or insufficient decontamination of flexible cystoscopes. Thus, high-level disinfection and sterilization are still needed to enhance cleaning efficacy. The current study demonstrated the feasibility of a combination of 405-nm laser and infrared (IR) lights to inactivate bacterial biofilms inside the working channels. Staphylococcus strains in suspension (concentration ~ 108 CFU/mL) were incubated in 24 hours at 37 0C to form biofilms on multi-well plates. Each sample initially experienced 5-minute IR dehydration (intensity = 1.5 W/cm2), and 405-nm laser system was then used for bacterial disinfection at various conditions: 19.1, 38.2, 57.3, and 76.4 J/cm2. An IR camera was deployed for real-time monitoring temperature on the biofilm surface. MTT assay was employed to assess cell viability after the treatment. The results showed that staphylococcus bacteria in liquid suspension were more resistant to 405-nm laser light than those in hard surfaces. In addition, the 405-nm laser treatment resulted in less decontaminating effectiveness while the 405-nm laser in conjunction with IR light could induce bacterial reduction up to 75% possibly due to effect low temperature dehydration. The proposed technique can be a useful modality for cystoscope cleaning and sterilization to prevent any secondary infection in the urinary tract during urological procedures. Further experiments will be conducted to validate reprocessing efficacy on various types of bacterial biofilm models along with high-level disinfectants to find the optimal cleaning doses.