The species diversity of culturable endophytic fungi was studied in the leaves and twigs of symptomatic and asymptomatic Fraxinus excelsior trees. Endophytic mycobiota was dominated by Ascomycota species, with Pleosporales (44.17%) and Diaporthales (23.79%) endophytes being the most frequently observed in the tree samples. The number of endophytic isolates and species richness varied depending on the sampling date (May and October) and tissue location. Of the 54 species identified based on ITS sequences, 14 were classified as dominant. The most frequently isolated species were Diaporthe eres, followed by Alternaria alternata, Dothiorella gregaria, and Fraxinicola fraxini. The inhibitory effect of 41 species (75 isolates) of endophytes on the radial growth of a Hymenoscyphus fraxineus isolate was studied under in vitro conditions (dual cultures). The radial growth of H. fraxineus was the most inhibited by four endophytic fungi from twigs (Fusarium lateritium, Didymella aliena, Didymella macrostoma, and Dothiorella gregaria). The inhibitory effect of the four isolates was also studied under in planta conditions. The isolates artificially inoculated into the trunks of ash trees reduced the length of necroses formed by H. fraxineus co-inoculated in the same trunks. This effect depended on the isolate, and the inhibition was most prominent only on trunks inoculated with F. lateritium and D. aliena. Although the total length of necrotic lesions formed by the H. fraxineus infection was shorter in the ash trunks co-inoculated with the endophytes, the difference was not significant.