Wildfires are a major problem in Mediterranean forest ecosystems, affecting the same area year after year. Their severity is increasing, partly due to climate change and hence, every now and then, virulent fires of high severity spread ravage this region. The aim of this study was to evaluate the influence of fire regime (recurrence, severity) in soil microbial community structure analyzing the phospholipid fatty acid (PLFA) and the microbial functional diversity assessing the level physiological profiling technique (CLPP). Samples of a soil affected by a high severities wildfire and a soil affected by a low severity experimental fire were heated under laboratory conditions at different temperatures to simulate different fire severity. To simulate fire recurrence, the heating treatment was repeated after one month of incubation. The fire severity was estimated as the amount of heat supplied to samples by degree-hour methodology. A marked impact of fire regime on soil microorganisms was detected; the microbial community response varied depending on previous history of fire and the magnitude of changes in PLFA pattern and CLPP, was related to the amount of heat supplied to the samples. Wildfires had a greater impact on microbial community structure than subsequent soil heating in the laboratory. The total biomass and the biomass of specific groups of microorganisms decreased notably as a consequence of wildfire and minor changes were detected due to the experimental fire and soil heating under laboratory conditions. The results clearly showed the usefulness of PLFA pattern to study the effect of fire regimes and associated direct and indirect changes in soil microorganisms and in soil quality. The data also indicated that the degree-hour methodology rather than maximum temperature is adequate to simulate fire severity and evaluate the impact of thermal shock on soil ecosystems.