We experimentally discriminated and quali-quantitatively characterized the extracellular fraction of a forest soil DNA pool. We sequentially extracted and classified the components of extracellular DNA by its strength of interaction with soil colloids as: (1) extractable in water, free in the extracellular soil environment or adsorbed on soil colloids; and as (2) extractable in alkaline buffer after previous extraction in water, bound on soil colloids. The comparative molecular analysis (fluorometer, gel electrophoresis, genetic fingerprinting) of directly and sequentially extracted extracellular DNA revealed quantitative and qualitative differences, also in terms of genetic information about microbial communities. The sequential extraction of extracellular DNA revealed differences in molecular weight, indicating a relationship between DNA fragment length and strength of interaction with soil colloids. The sequential extraction was also suitable to assess the presence of tightly bound DNA, providing information about the DNA-colloid interactions naturally occurring in the soil environment.