Grapevine is one of the most important fruit trees which has been used by human since ancient times and is considered as a major agricultural product in Iran. However, Vitis vinifera (the cultivated species) is susceptible to many fungal pathogens which can lead to economic losses or decline of the vineyards. Among them, grapevine trunk diseases (GTDs) are problematical and associated with many different fungi, such as Cytospora chrysosperma. 293 endophytic and GTDs-associated fungi were isolated from 7 provinces of Iran. Based on morphological characteristics, 25 representative isolates were selected and identified using internal transcribed spacer (ITS-rDNA) region. They belonged to 14 genera of Aspergillus, Cephalotrichum, Chaetomium, Coniochatea, Fusarium, Microascus, Neosetophoma, Penicillium, Phaeoacremonium, Phialemonium, Preussia, Stromatinia, Tricharina, and Truncatella. Using the dual culture method, the antagonistic activity of 13 isolates from those 25 selected ones was shown against Cytospora chrysosperma; in which its hyphal growth was inhibited from 10 to 41.33%. Among tested fungi, Fusarium sp3.-GI154, Fusarium sp4.-GI267, Fusarium sp5.-GI289, Penicillium canescens-GI101 and Chaetomium sp.-GI179 showed the highest antifungal activity, respectively. Peptide exctract of Fusarium sp.3-GI154 displayed inhibitory activity against Cytospora chrysosperma. To the best of our knowledge, this is the first report of Aspergillus europaeus, Cephalotrichum verucisporum, Microascus chartarus, Neosetophoma salicis, Penicillium canescens, Penicillium expansum, Preussia minima and Tricharina sp. as endophytes and Phialemonium sp. as a GTDs-associated fungus from grapevine in Iran. Endophytes can provide an interesting field for searching appropriate approaches of grapevine diseases biological control.