DNA extraction from macrofungi is a prerequisite for all the downstream biotechnology-derived applications such as Sanger and genome sequencing, microarrays. However, extraction of high-quality DNA from macro-fungal tissues is often painstaking, challenging, and may pose a delay for high-throughput experiments, such as DNA sequence identification based biodiversity, invasion or impact assessment surveys. Commercial DNA extraction kits often prove to be either expensive or inconsistent in DNA extraction from diverse macro-fungal species samples, such as mushroom genera, where fruiting bodies vary greatly in texture, size, chemical composition and pigmentation. The present study implemented Dimethyl Sulphoxide (DMSO) to lyse cell walls of dried, grounded fungal samples for DNA extraction. The technique is efficient, rapid, and affordable and requires a low amount from the sample for DNA extraction. Furthermore, both the quantitative and qualitative data demonstrate superior grade DNA that yield products in subsequent PCR amplification of the ITS-5.8S phylogenetic marker region. This technique will allow researchers from underdeveloped and developing countries with varying levels of expertise to extract mushroom DNA even with a low research budget.  It will also enable biodiversity, conservation, impact assessment, and invasion biology surveys in these countries, as well as fungorium based research.