Microbial diversity plays a crucial role in ecosystem processes, including organic matter decomposition and nutrient cycling. This research explores the effect of prescribed burning (PB) on soil microbial diversity, as well as biomass and activity in Mediterranean pine plantations. In burned and adjacent unburned plots of Pinus pinea and P. pinaster plantations of Southern Italy protected areas, the fermentation layer and the 5 cm thick layer of mineral soil underneath were sampled at intervals during the first year after PB. The experimental protocol encompassed measurements of total microbial abundance (Cmic and soil DNA), fungal mycelium, fungal fraction of Cmic, microbial activity, bacterial genetic diversity (16S rDNA PCR-DGGE), microbial metabolic quotient (qCO2), and C mineralization rate (CMR), as well as physical and chemical soil properties. PB caused only temporary (up to 3 h–32 d) reductions in Cmic, DNA amount, fungal mycelium, respiration, and CMR in the P. pinaster plantation, and had no appreciable negative effect on the microbial community in P. pinea plantation, where fire intensity was lower because of less abundant litter fuel. In either plantation, PB did not generally reduce bacterial genetic diversity (evaluated as band richness, Shannon index, and evenness), thus, also accounting for the fast recovery in microbial growth and activity after high-intensity PB in P. pinaster plantation. While confirming PB as a sustainable practice to reduce wildfire risk, also supported by data on plant community obtained in the same plantations, the results suggest that an integrated analysis of microbial diversity, growth, and activity is essential for an accurate description of PB effects on soil microbial communities.