Complex procedures are required for DNA extraction from filamentous fungi owing to the structural complexity of their cell walls. Disruption of fungal cells results in the release of large amounts of polysaccharides and proteins. The objective of the present study was to improve the yield and purity of DNA through the development of a refined protocol for extracting dermatophyte genomic DNA from the culture medium and to standardize each step of the extraction procedure. Different culture media compositions (dextrose/maltose) were compared to assess fungal growth. The initial biomass was standardized, and the mycelial biomass was subjected to liquid nitrogen grinding. A modified phenol-chloroform extraction method was used with a five-step washing procedure. Genomic DNA was subjected to quantitative and qualitative analyses by spectrophotometry and 1% agarose gel electrophoresis, respectively. The average amount of DNA obtained in the study from Sabouraud’s dextrose broth with Tween 80 was 1218.5 ng/µL. Amplification was performed using internal transcribed spacer primers. The A260/A280 ratios ranged from 1.7 and 2.0. The genomic DNA obtained had a sufficient molecular weight (>1,000 kb). Successful amplification with ITS primers designed for fungal DNA confirmed sufficient removal of PCR inhibitors during both fungal cell preparation and DNA extraction. The standardized, improved protocol for dermatophyte DNA provides a useful guideline for mycology researchers to work with fungal genomic DNA of sufficient molecular weight, which is best suited for downstream processes such as PCR, sequencing, and marker-based phylogenetic studies.