Objective: Healthcare-associated infections due to Gram-negative bacteria (GNB) are a major cause of mortality and morbidity throughout the world. The purpose of the research described here was to evaluate the possibility of using an extremely low-concentration gaseous chlorine dioxide (ClO 2 , 0.01 ppmv, 0.028 mg/m 3) as a technique to reduce the risk of environmental infection by GNB. In this study we set up an exposure chamber (1 m 3) and used three types of GNB, namely Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii. Results: The extremely low-concentration gaseous ClO 2 inactivated E. coli (> 2 log 10 reductions, within 2 h), P. aeruginosa (> 4 log 10 reductions, within 2 h) and A. baumannii (> 2 log 10 reductions, within 3 h) in wet conditions on glass dishes. Treatment of moist environments with extremely low-concentration gaseous ClO 2 may help to reduce the risk of environmental infection by GNB without harmful effects.