Aims: To determine a temperature-humidity-time treatment that eliminates Salmonella and Escherichia coli in substrates representing organic matter in poorly cleaned poultry houses, i.e. worst case scenario laboratory tests. Methods and Results: Organic matter (poultry faeces and feed) in a 2.5-cm layer was inoculated with 2x10(5) -3x10(6) Salmonella g(-1) , left undried or dried at ca. 30% relative humidity (RH) during a 10-day period, and temperature increased at 1degreesC h(-1) to the final heating temperature of 50, 55, 60, 65 or 70degreesC and held at 16-30 or 100% RH. All samples were tested for Salmonella according to predetermined sampling time schedules and faecal samples were also tested for naturally occurring E. coli . Overall, humidity was an important factor in the elimination of Salmonella and E. coli . Results for recovery of Salmonella and E. coli were highly associated. Conclusions: The application of greater than or equal to60degreesC and 100% RH during a 24-h period eliminated Salmonella and E. coli in all samples. Escherichia coli could be used as an indicator bacterium for the elimination of Salmonella . Significance and Impact of the Study: The results from worst case scenario laboratory tests could be applied in steam heating of persistently Salmonella -infected poultry houses. The use of E. coli as an indicator bacterium for the validation of Salmonella results should be considered.