Extraction of pure genomic DNA from fungi such as Candida sp., Saccharomyces sp./Cryptococcus sp. and molds like Fusarium sp. is essential for a wide range of molecular biology research applications, such as polymerase chain reaction (PCR), genotyping and sequencing. These different species present varied challenges in DNA extraction, particularly due to structural differences in their cell walls. In particular, Cryptococcus sp. possesses a thick polysaccharide capsule that must be effectively removed during the extraction process, requiring a more rigorous approach to ensure complete lysis. In contrast, other fungi such as Candida sp./Saccharomyces sp. do not have capsules, so their extraction is relatively simple and primarily requires mechanical disruption and cell lysis buffer for efficient cell wall degradation (Tapia et al, Mol Biotechnol 33(1):67–70 2010). However, molds such as Fusarium sp., pose an additional challenge due to their rigid and multilayered chitinous cell wall, which is often thicker and more resistant than yeasts. These complex cell wall components require a more aggressive lysis approach to achieve complete cell wall degradation. Our protocols utilize mechanical disruption by bead beating, which applies sufficient force to break down the robust cell wall structure, followed by the application of lysis buffers designed to degrade both cell wall components and intracellular proteins. This technique is founded on the phase separation principle, in which phenol-chloroform is used to separate lipids and proteins in the organic phase and isolate nucleic acids in the aqueous phase. To obtain high-quality genomic DNA, the extraction protocol includes precipitation steps with ethanol or isopropanol, effectively concentrating the DNA, while removing impurities. This optimized approach ensures efficient recovery of high-quality genomic DNA and significantly increases the reliability of downstream analyses (Codreanu, Ciurea, Microorganisms 11(4):818, 2023; Bolano et al, FEMS Yeast Res 1(3):221–224, 2001).