Flexible endoscope reprocessing is an important requirement to minimize the risk of cross-infection between patients due to incomplete disinfection of a bacteria biofilm. The present study introduces a novel opto-chemical treatment to disinfect microbial biofilms (both Gram-positive and Gram-negative bacterial biofilms), commonly found in flexible endoscopes. A low concentration disinfectant combined with infrared and blue light irradiations was applied to disinfect the bacterial biofilms in the endoscope. A basket-integrated optical device was designed to deliver uniform and concentric light onto the channel surface of the endoscope. Colony-forming unit assays were performed to quantify the vial cells while scanning electron microscopy (SEM) illustrated an extracellular matrix (ECM) of the bacterial biofilm. The infrared light irradiation heated the surface of the bacterial biofilm to ~ 65oC. The blue light irradiation induced a relative temperature increase of 30oC on the bacterial biofilm. The results showed that the opto-chemical treatment reduced approximately 7.5-log10 of the bacterial biofilm, which was four times higher than that of a standard disinfectant solution (2% glutaraldehyde). In comparison with the control untreated samples with intact ECMs, the SEM images showed significant damage to the bacterial biofilm under the opto-chemical treatment. The combined treatment induced antimicrobial effects in terms of inhibition of protein synthesis, thermal destruction, and oxidative stress, eradicating the bacterial biofilm more than the standard chemical disinfection. The proposed technique could be an alternative approach to disinfect the microbial biofilms and minimize the risk of secondary infection in endoscopy-related medical facilities.