The objective of this study was to determine the influence of relative humidity (RH) on the antimicrobial effect of chlorine dioxide (ClO 2 ) gas against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes on spinach leaves. Spinach leaves were inoculated with three foodborne pathogens and treated with ClO 2 gas at different concentrations (1, 5, 10, 30, and 50 ppmv) for 0, 5, 10, 15, and 20 min under differing conditions of RH (50, 70, and 90%). Inoculated spinach leaves subjected to ClO 2 gas treatments experienced significant reductions, with pathogen populations reduced more under conditions of 90% than under 50 and 70% RH. Generally, there were no significant differences (p > 0.05) in reduction levels of the three foodborne pathogens between 50 and 70% RH. Exposure to 50 ppmv of ClO 2 gas for 20 min resulted in 1.25–1.78 (50% RH) and 2.02 to 2.54 (70% RH) log reductions of the three foodborne pathogens. The levels of the three foodborne pathogens was reduced to below the detection limit (1 log CFU/g) within 15 min when treated with 50 ppmv of ClO 2 gas under 90% RH. The results may help the fresh produce industry to establish effective conditions for ClO 2 gas treatment.