The soil DNA pool consists of an intracellular (iDNA) and extracellular fraction (eDNA). Challenging to improve the extraction efficiency of soil DNA, and to quantitatively and qualitatively characterize both DNA fractions, we set up a molecular approach consisting of sequential and comparative DNA extraction and microbial community fingerprinting. eDNA was extracted by alkaline soil washings (ASW); iDNA by mechanical chemical cell lysis (MCCL) of the residual soil pellet after the extraction of eDNA (ASW–MCCL). The molecular approach was compared in terms of quantity (fluorometer) and quality (agarose gel electrophoresis; small subunit rRNA-denaturing gradient gel electrophoresis) to directly extracted soil DNA comprising both eDNA and iDNA (tDNA; FastDNA Spin Kit for Soil, BIO101). The proposed method appeared to be a potential tool to separately extract and analyze eDNA (6.07 μg g −1 soil) and iDNA (11.46 μg g −1 soil) and to obtain a greater amount of DNA from soil with broader genetic information about eubacterial and fungal communities with respect to directly extracted tDNA (8.79 μg g −1 soil). Our results revealed the extracellular fraction to be quantitatively and qualitatively important of the soil metagenome. As the sequential DNA extraction method not only increased the total amount of extractable soil DNA (17.53 μg g −1 soil) but also that of iDNA, it is suggested to be suitable for extracting the soil metagenome.