Medical societies and public health agencies rigorously emphasize the importance of adequate disinfection of flexible endoscopes. The aim of this work was to propose a novel opto-chemical disinfection treatment against Staphylococcus aureus grown in mature biofilm on Teflon-based endoscope channel models. Laser irradiation using near-infrared and blue wavelengths combined with a low concentration of chemical disinfectant induced both irreversible thermal denaturation and intercellular oxidative stress as a combined mechanism for an augmented antimicrobial effect. The opto-chemical method yielded a 6.7-log10 reduction of the mature Staphylococcus aureus biofilms (i.e., approximately 1.0-log10 higher than current requirement of standard treatment). The proposed technique may be a feasible disinfection method for mitigating the risk associated with infection transmission.