Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 microg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences.