Aims The development of high-throughput methods such as pyrosequencing and microarrays has greatly improved our understanding of the microbial diversity in complex environments such as soils. Nevertheless, albeit advancements in such techniques, the first major step is to obtain high quantity and good quality genomic DNA (gDNA). The work presented here aims to present an inherent problem with 260:230nm ratio of extracted gDNA from calcareous soils of Tuber melanosporum orchards and a protocol to overcome this problem. Methods and Results Using two commercial gDNA extraction kits on spatially distant truffle orchards, we demonstrated that the 260:230nm ratio was very low, consequentially yielding gDNA incompatible with microarray analyses. In order to solve this problem, optimization steps were tested including several wash steps performed before and/or after lysis. These washes significantly improved the gDNA quality (ratio 260:230nm >1 center dot 7) without modification of the structure of the bacterial communities as stated by temporal temperature gradient gel electrophoresis analysis. A final re-extraction with phenol/chloroform was required for one of the soil samples. Conclusions A combination of wash steps included into the extraction protocol followed by phenol: chloroform re-extraction is recommended to obtain high-quality gDNA from calcareous soils of T.melanosporum orchards. Significance and Impact of the Study The method recommended here significantly improves gDNA quality obtained from T.melanosporum orchards to make it acceptable for highly sensitive methods such as microarray.