Five commercial DNA extraction kits for microbial DNA in soil were evaluated in terms of the extraction efficiencies of artificially-spiked extracellular DNA in soil. Commercially-available purified DNA derived from the sperm of Clupea harengus (Atlantic herring) was homogeneously premixed in blank soil samples and extracted using the kits. The spiked DNA in each of the extracts was quantitated by real-time PCR to evaluate extraction efficiency using a specific primer set for the mitochondrial 16S rRNA gene of Clupea harengus . Only the Extrap Soil DNA Kit could extract the DNA from all types of soils spiked at 10 μg/g soil (vegetable garden soil 0.85%, courtyard soil 0.30% and virgin andosol 0.027%), whereas other kits (Nucleospin Soil, Power Soil DNA Isolation Kit, ISOIL and ISOIL for Beads Beating) could not extract the DNA larger than the quantitation limit (0.020%) from the courtyard soil and the virgin andosol which exhibited high phosphate absorption coefficient. Skim milk concentration and bead-beating time using the Extrap Soil DNA Kit were optimized for 20 mg/g soil and 30 s, respectively. Significant PCR inhibition was not observed under the optimized condition.