DEAD-IN-SHELL embryos is represented the main cause of ……  hatchery losses in poultry. There is limited literature about the role of fungi in the hatchability problems. In this study, different fungi were isolated from dead-in-shell embryos of local Egyptian breeds. Some randomly selected isolates were molecularly identified, and the antifungal activity of extracted essential oils was investigated on these isolates. Seventy nine fungal isolates were recovered in the rate of 66.9% of which A. fumigatus, A. niger, Cladosporium spp. and Penicillium Spp. were represented in a prevalence of 28.0, 18.6, 11.9 and 8.5% respectively. Aspergillus spp. remains the highest prevalent fungi that recovered from dead-in-shell embryos among chicken and Turkey embryos. The cinnamon and thyme oil are the superior antifungals extract with concentrations as low as 0.05%, followed by clove oil. However, high concentrations of rosemary (up to 0.5%) are needed to inhibit the fungal growth. Fungal isolates were successfully characterized using PCR employing specific primers for the ITS region and β-tubulin gene of Aspergillus spp. In conclusion, these results indicated the importance of Aspergillus spp. in hatchability problems. PCR detection was proven to be the more rapid and the simplest tool for identification of Aspergillus spp. Moreover, essential oils can be used as efficient antifungals.