Successful detection and characterization of microbacterial DNA in the environment require efficient extraction of the DNA from environmental samples and adequate purification from the coextracted contaminants that inhibit PCR. Previously reported methods for direct isolation and purification of DNA from microorganisms in soil have multiple steps, laboratory equipment’s and labour-intensive, and result in loss of extracted material. This has promoted the initiative to develop an efficient, nonselective method for direct DNA extraction from the environment that is required for rapid and thorough analysis of introduced microorganisms in environmental samples and for diversity analysis of microbial community in soil. The newly developed method is based on the combination of hot-detergent treatment and commercial spin columns. Fidelity and effectiveness of the procedure were investigated by identifying several phylogenetic groups representing different components of the soil microbial community. In addition, the use of HotStar Taq DNA polymerase has provided the opportunity for simultaneous detection of a variety of microorganisms in a single tube, using multiplex PCR. The ability to simultaneously extract and amplify DNA from many types of microorganisms using a simple extraction and purification step is desirable for many applications in molecular microbial ecology.