Beauveria bassiana, a necrotrophic fungus thriving in diverse ecosystems, predominantly on arthropods, holds significant importance as a biological agent. The escalating relevance of genetic studies on fungi necessitates the development of efficient and robust DNA extraction techniques to ensure the acquisition of high-quality DNA. B. bassiana was cultured to produce either mycelia or blastospores. Six DNA extraction procedures and two pre-treatment conditions were independently adopted to evaluate their efficacy in obtaining high molecular weight, high concentration, and high-quality DNA from both blastospores and mycelia. Quantification of the extracted DNA was achieved through qPCR analysis targeting the single-copy γ-Actin gene, with validation of amplification specificity and determination of amplification efficiency conducted through standardized protocols and standard curve construction. Statistical analysis indicated that, under all conditions, blastospores yielded more promising results. Blastospores exhibited a mean antilog of 5.0344 + 0.20 for DNA copy concentration, surpassing mycelial cells averaging 2.5069 + 0.20. Additionally, blastospores outperformed mycelial cells in DNA quality, as evidenced by their 260/280 and 260/230 ratios (31.5% within the range for blastospores vs. 7.4% for mycelial cells, p-value=0.002). Moreover, blastospores produced higher molecular weight DNA, averaging 10770.5 base pairs compared to mycelial cells at 8139.0 base pairs (p-value <0.001). This study underscores the superiority of DNA extraction from blastospores compared to mycelial cells of B. bassiana. These findings have broader implications for other dimorphic fungi and open new avenues in the realms of molecular biology and microbial ecology, particularly in genetic and evolutionary studies.