Background: Infections caused by pathogenic fungi have increased in recent years and well recognized to be one of the important causes of death especially in immunocompromised patients. The diagnosis of fungal infections remains a significant problem, mainly because cultures are slow-growing and identification is laborious and difficult, especially for moulds. However, rapid identification of pathogenic fungi is essential to start antifungal treatment. Conventional identification of moulds is mainly performed by phenotypic criteria, including micro- and macromorphology. Phenotypic identification procedures are time consuming and require expertise in fungal morphology. However, specialists and expertise in medical mycology field are decreasing due to several reasons, requiring the application of easy genetic assays such as PCR-sequencing, PCR-RFLP and PCR-fingerprinting. These techniques are successfully applied to identify pathogenic fungal isolates (moulds and Yeasts). Methods: We previously identified 70 fungal strains by conventional methods such as culture, microscopy and biochemical tests. Then, DNA was isolated from pure cultures of fungi by simple and rapid chemical (phenol: chloroform: isoamylmalcohol) based DNA extraction protocol. PCR method for identification of pathogenic fungi based on the amplification of the Internal Transcribed Spacer (ITS1/2) region was used. After the PCR products were sequenced, GenBank Blast searches were performed, and the sequence-based and conventional identifications were compared. We also decided to identify our fungal strains by using ITS-RFLP and PCR-fingerprinting. Results: In this study, our data showed that we were able to identify 13 mould strains up to the species level and 7 mould strains up to the genus level. 20 different species of moulds were identified by conventional techniques; the most common were dermatophytes (42.2%) followed by Aspergillus species (40%) and some other miscellaneous moulds (18.8%). 45 mould strains belonged to 20 genera investigated in this study included 12 Aspergillus niger and 9 dermatophyte isolates. 13.3% of the total number of moulds studied were not identified to the species level. The most frequent identifications of 25 strains of yeasts were as following: Candida tropicalis (9/25), followed by Candida albicans (7/25), Candida glabrata (4/25), Geotrichum capitatum (3/25) and Candida parapsilosis (2/25). By contrast, 65 fungal strain were identified by PCR- sequencing and showed that 64 (98.4%) identified to species level and 1 (1.6%) to genus level. Also PCR-RFLP showed (100%) similarity to the identification by ITS sequencing and (82.9%) with culture results. There was 100% agreement between the PCR-RFLP analyses and PCR-fingerprinting analyses for the identifications obtained of the studied Candida species. Conclusions: Conventional methods of identification of fungi have mainly relied on culture isolation and the observations of morphological features. These methods are time consuming, laborious, and may require days to weeks for the identification by culture. PCR of the ITS region followed by sequencing can be used to identify pathogenic fungal organisms in pure culture but it is expensive. Analysis of the ITSRFLP can be an alternative tool for a fast and reliable identification of fungi in microbiological laboratories. In addition, PCR-fingerprinting analysis is not only faster and simple to use, but useful given its consistency with conventional methods.