We investigated microbiota in surface and subsurface soil from a site, above steam-treated deep sub-soil originally contaminated with chlorinated solvents. During the steam treatment, the surface soil reached temperatures c. 30°C higher than the temperature in untreated soil; whereas the subsurface soil, at a depth of about 40 cm, reached a temperature c. 45°C higher than untreated soil. The soil was examined prior to, during, and 6, 12, 14, 20 and 31 months after treatment. Numbers of bacteria cultivable at 42°C increased significantly in subsurface soil. Similarly, substrate utilization in ECOLOG plates, incubated at 42°C, increased from less than 10% of available carbon sources in the untreated soil to more than 60% of the available carbon sources in the steam-treated soil. Aspergillus fumigatus was quantified as an example of heat-tolerant fungi normally found in compost. These organisms are rarely detected in Danish soils but high numbers (c. 105 hyphal forming units g−1) occurred in the treated soil up to 31 months after the steam-treatment. We conclude that steam-treatment leads to changes of the microbial communities. Some changes are temporary while others can last for years after termination of the steam-treatment; reflecting different strategies that soil microorganisms follow.