The aim this study was compare and evaluate the suitability of the use of phenotypic and molecular methods in the identification of microscopic filamentous fungi (MFF). We subjected 329 isolates of MFF isolated from chicken eggs to phenotypic and genotypic identification. In this study, we used and compared three pre-isolation procedures for fungal DNA isolation: 1. combination of liquid nitrogen and heat 95 °C, 2. proteinase K and FG1 lysis solution, and 3. isolation using zirconium and glass beads with simultaneous effects of proteinase K and ultrasound (50 Hz). For genotypic identification, we used the method of conventional polymerase chain reaction (PCR), where we designed species-specific primer pairs VERF / VERR, COMF / COMR, CRUF / CRUR and Pvi-F / Pvi-R based on the 5.8S-ITS region for identification of four Penicillium species (P. verrucosum, P. commune, P. crustosum and P. viridicatum). We confirmed the results of the conventional PCR method by PCR-ITS-RFLP method, where we created ribosomal restriction patterns of the amplified ITS regions using seven restriction endonucleases (Bsp1286I, Xmal, HaeIII, Hinfl, MseI, SfcI, Hpy188I). The pre-isolation procedure using zirconium and glass beads, proteinase K and ultrasonic waves was proved to be the most effective and was also sufficient for isolates of Cladosporium spp., where other isolation methods did not give satisfactory results. Using phenotypic methods, coventional PCR method and PCR-ITS-RFLP method, we identified isolates as Alternaria alternata (19/17/17), Cladosporium cladosporioides (64/64/64), Penicillium verrucosum (12/8/8), Penicillium commune (48/36/36), Penicillium crustosum (21/19/19), Penicillum viridicatum (35/31/32), Penicillium chrysogenum (13/13/13), Aspergillus fumigatus (53/53/53), Aspergillus sydowii (9/8/9), Fusarium proliferatum (23/21/20) and Fusarium verticillioides (11/10/11). The results of this study showed the lowest coincidence in the identification methods in the group of Penicillium spp. The PCR methods used in our study demonstrated the ability to differentiate MFF species isolated from chicken eggs. Based on efficiency, conventional PCR appears to be more advantageous compared to PCR-ITS-RFLP because it is less time and money consuming.