A new manual on infection control of otolaryngological endoscopy has been published, in which chlorine dioxide is described as a second-line disinfectant after peracetic acid, orthophthalaldehyde and glutaraldehyde. Chlorine dioxide itself is not recognized as a high-level disinfectant, but its use for disinfecting gastrointestinal endoscopes is approved by the Japanese government. In this study, we investigated bacterial contamination of gastrointestinal endoscopes before/after disinfecting with chlorine dioxide. In 65 cases of upper gastrointestinal endoscopy and 60 cases of colonoscopy, 10ml of saline was injected into the channel of the endoscope and the washing solution was collected into culture media, before/after cleansing. Bacteria were identified in all cases before cleansing. In contrast, all of the upper gastrointestinal endoscopes and 59 of the 60 colonoscopes were negative for bacteria after cleansing. Only one case of colonoscope was positive for E. coli by enrichment culture. No damages to the endoscopic instruments were caused and no adverse events related to the skin and lungs of medical staff occurred in the endoscopy room. The cleansing and disinfecting device with chlorine dioxide was effective in eradicating bacteria on gastrointestinal endoscopes.