Escherichia coli and Salmonella spp. are considered to be the major pathogens associated with human transmissible infectious diseases in the air of poultry houses. Chlorine dioxide (ClO2) is an effective biocide against a wide range of microorganisms. Accordingly, this study investigated the efficiency of gaseous ClO2 application for disinfecting broiler houses by collecting air samples before and after fumigation using a passive method. Fumigation was performed with 125 mL or 250 mL of ClO2 liquid (containing 2,000 ppm of ClO2) and 3 trials were conducted for each dose. A total of 27 petri dishes were used for each trial (for each type of bacteria: E. coli or Salmonella) and placed in 3 different locations (front, middle and back) and 3 different positions (top, middle and floor) of the broiler shed. Air samples were collected at 10 min, 1 h, 3 h, 6 h, and 12 h before and after fumigation to evaluate the air quality in terms of the concentration of E. coli and Salmonella. Both levels of ClO2 were capable of reducing the concentration of E. coli from broiler house air during all measuring periods except 10 min, with highest disinfection rate being observed at 6 h. With the exception of 1 h, the concentration of Salmonella was also reduced after fumigation with ClO2 in all measuring period; with the highest disinfection rate occurring at 6 h. Fumigation with ClO2 had no negative effect on birds' health condition. Taken together, these results suggest that the application of gaseous ClO2 at the investigated levels can be an effective option for reducing bacterial load from broiler house environments.