Fungal endophytes were isolated from the Mediterranean cypress Cupressus sempervirens. Eleven taxa of fungi, all within the Ascomycota, were identified based on PCR amplification and sequencing of the internal transcribed spacer sequences of nuclear ribosomal DNA (ITS rDNA) with taxonomic identity assigned using the NCBI nucleotide megablast search tool. The endophytic fungi included Alternaria multiformis, Didymella sp., Phoma sp., Phoma herbarum, Pyrenochaeta sp. (Dothideomycetes), Penicillium brevicompactum, Talaromyces sp. (Eurotiomycetes), Ascorhizoctonia sp. (Pezizomycetes), Thielavia microspora, and Thielavia spp. (Sordariomycetes). Considering the former findings in US, this indicates that similar ascomycetous classes of fungi, all from Pezizomycotina, associate with the healthy Cupressaceous trees in Iran. The recovered endophytes produced antifungal and antiproliferative metabolites which may contribute to the protection and survival of the host. We speculate that endophyte-infected C. sempervirens may benefit from their fungal associates by their influence on the ecology and biotic stress tolerance of the host plant. Moreover, a novel niche for the identified fungal species is being introduced.