There are many methods for identification of yeasts and dermatophytes including phenotypic and PCR methods. Recently, FT-IR spectroscopy is increasingly being used for identification of yeasts and dermatophytes. This study aimed to investigate the capability of this technique to identify and discriminate between the most frequent yeast and dermatophytes species isolated from human and animals. To that end, yeast and dermatophytes isolates were identified by phenotypic method, RFLP, RAPD PCR and FTIR spectroscopy analysis. A total of 166 isolates (128 yeasts and 38 dermatophytes) were isolated from 266 samples represented in: 75samples from human cases (20 vaginal , 20 oral swabs and 35 skin scrapings , hair and nail scrapings), chronic diseased cows (131 sample including 21 rectal, 20 vaginal, 26 oral, 19 nasal, 20 ear swabs and 20 mastitic milk in addition to 5 skin scrapings from cattle ringworm), chicken crop mycosis (20 swabs) and five cases of dog dermatophytosis. In addition to, thirty five soil samples including 25 soil contaminated with pigeon droppings for isolation of C.neoformans and 10 soil samples for isolation of geophilic dermatophytes. The present work revealed that, while genotypic method and FTIR could identify yeast and dermatophytes into levels of genera, species and even to varieties. Phenotypic methods not enough to identify most yeast species and dermatophyte varieties. Phenotypic identification of yeasts isolates revealed that, the most Candida spp. isolated from human oral and vaginal candidosis was C.albicans (30%) followed by C. tropicalis (5%) then C.parapsilosis and C.glabrata (2.5%) for each. Concerning cow samples, 53 Candida isolates were obtained. The most Candida spp. were C. krusei (35.8%) followed by C. albicans (28.3%) then C. parapsilosis (11.3%), C.glabrata (7.5%) and C.tropicalis (5.6%). From mastitic milk samples C.lipolytica, C.vini, C. inconspicua, C. peltata, C.albicans and R.rubra were isolated. C.albicans (60%) was the most Candida spp. isolated from crop mycosis followed by C.glabrata (20%) then C.krusei (10%) and C.lusitaniae (10%). Five isolates (20%) of C.neoformans, 4 Trichosporon spp. (16%), 9 G. candidum, 4 R.rubra, 2 C.kefyr and one isolate C.saitoana were obtained from soil contaminated with pigeon droppings. Concerning with genotypic identification, PCR-RFLP was applied for identification of Candida spp., Cryptococcus neoformans and Trichosporon spp. using universal primers ITS1 and ITS4 amplified ITS region. And it yielded a unique PCR product size of approximately 358-871 bp. PCR amplicons were digested with enzyme Msp1 and the generated bands corresponded to the predicted sizes. Phenotypic identification of dermatophyte isolates revealed that, Six different species were recovered from human nail, skin scrapings and hair were identified in to T.violaceum (36%), M.canis (29%), T.rubrum (18%), T.mentagrophytes (11%), E.floccosum (4%) and T.verrucosum ( 4%). All isolates obtained from cattle were identified as T.verrucosum (3) while those isolated from dogs were identified as M.canis (2) and T.verrucosum ( 1 ). Using hair bait technique for ten soil samples , four M.gypseum isolates (40%) were obtained. Genotypic identification of dermatophytes revealed that, it gave good identification for confused species and it identify T.mentagrophytes into varieties level. Eighteen dermatophyte isolates were identified by two PCR-based methods. ITS based PCR method and Arbitrarily primed PCR (AP-PCR). Using common primer ITS1\ITS4 primer set resulting in amplified products of approximately 690 bp in T.mentagrophytes, T.rubrum , T.violaceum and T.verrucosum, M.canis at 740 bp, M.cookei at 600 bp and M.gypseum at 640bp. Using AP-PCR technique with random primer OPD18, dermatophyte isolates could be distinguished on the basis of characteristic band patterns detected in agarose gel electrophoresis. Into T.mentagrophytes var nodular, T.mentagrophytes var quinckeanum, T.rubrum, M.canis, M.cookei, M.gypseum and T.verrucosum. Concerning identification using FTIR spectroscopy, FTIR Spectra of yeasts and dermatophytes of different genera and species which fully identified by phenotypic and PCR identification were used as a private library data base (reference for the different species). Spectra of different samples from the same species showed great similarity and matching (100%) with the original library data base. When thirteen unknown tested samples were identified by FTIR spectroscopy and their spectra were compared with reference curves, they looked identical. Hierarchical cluster analysis (HCA) was applied for grouping the spectra with the same degree of similarity. The merging process visualized in a dendrogram, presenting the regrouping of the spectra in clusters according to a heterogeneity scale. Antifungal sensitivity test using disc diffusion method for yeast isolates recorded that, while Candida and Cryptococcus species were sensitive to ketoconazole, fluconazole. Trichosporon species were sensitive to clotrimazole and amphotericin B. Geotrichum spp. showed higher susceptibility to ketoconazole. Rhodotorula spp. showed higher susceptibility to nystatin. As regards the antifungal sensitivity of dermatophyte species, terbinafine and griseofulvin were effective in inhibiting most species followed by itraconazole and fluconazole.