A set of chemical, physical and microbial characteristics of different burnt soils from Sierra de Aznalcóllar (Sevilla, Spain) affected by one or two sequential fires, were analysed and compared with those of their respective control soils. A decrease in total organic carbon was observed in burnt soils, which could be attributed to the impact of the fires on vegetation cover. Biomass (estimated as viable and culturable microorganisms), substrate-induced respiration (SIR) and activity of different soil enzymes involved in carbon, nitrogen and phosphorus cycles were determined to assess the effect of fire on total microbial populations and on soil activity. An increase in both bacterial and fungal biomass as well as respiratory activity was detected in most burnt soils. In terms of enzyme activity, no common pattern of behaviour was observed, except for the alkaline phosphatase activity, which showed increased levels in all the burnt soils. The effect of fire on microbial diversity was estimated for Bacteria and Archaea domains from DNA band patterns obtained in denaturing gradient gel electrophoresis (DGGE), as well as using 16S rRNA cloned sequences for Bacteria. Shannon index values obtained from the DGGE profiles showed higher diversity for both Bacteria and Archaea domains in burnt soils compared with the control ones. Variations in the number of different phyla present in burnt and control soils were inferred from the analysis of the 16S rRNA cloned sequences. However, in all areas the most important groups identified belonged to the Proteobacteria, Acidobacteria and Actinobacteria phyla. No differences between microbial communities present in burnt soils at the genus level were detected.