Infections caused by opportunistic fungi are a significant health problem in immunocompromised patients. Rapid identification and characterization of these fungi is of high importance in timely initiation of antifungal treatment [1]. Currently fungal characterization is performed by classical culture based assays, which are laborious, time-consuming and require detailed knowledge of micro- and macroscopic morphology of fungi [2]. To solve these problems application of molecular based methods, like, PCR, have been recommended, however, problems are often encountered in terms of relatively low DNA yields and purity even with commercially available DNA extraction products [3]. Therefore, the development of an appropriate method for efficient DNA extraction from fungi is still needed. In the present study, a laboratory modified chemical 1% SDS-lysis method and three commercial available kits (GeneJET Genomic DNA Purification Kit, QIAamp DNA Mini Kit and NucleoSpin Plant II Kit) were examined to determine the most appropriate DNA isolation method for Fusarium solani detection and enumeration. The quality and quantity of the differentially extracted DNA was compared by spectrophotometric measurements and real-time PCR amplifications. The results of mean purity ratio of 260/280 nm for all commercially available extraction kits were below 1.6 or greater than 2.00 indicated that these tested kits were not appropriate for F. solani DNA isolation. The SDS-lysis protocol with two-membrane filtration step modification proved to be the most efficient DNA extraction method and might be adapted for the extraction of fungal DNA for further sample analysis with real-time PCR.