Fraxinus angustifolia, the main ash species in Croatia in terms of economic and ecological importance, is affected by a severe dieback initially attributed to the fungal pathogen Hymenoscyphus fraxineus. Recently, another pathogen, Diplodia fraxini, has been shown to play a key role in ash dieback in several European countries. Therefore, because the dieback symptoms of ash trees observed in Croatia are typical of Botryosphaeriaceae attacks, the aim of this study was to define the etiology of F. angustifolia dieback. To this end, symptomatic shoots and branches and cross-sections of the main stem were sampled from 20 symptomatic trees at eight locations and analyzed for the presence of D. fraxini and other possible fungal pathogens. Diplodia fraxini was the fungus most frequently associated with branch cankers and dieback; it was isolated from 17 trees in all sites monitored, and its pathogenicity towards F. angustifolia was confirmed. The fungus was also associated with wood necrosis at the base of the trunk in two trees. Other fungi, namely H. fraxineus, Diaporthe eres, Diplodia seriata, Botryosphaeria dothidea, Armillaria gallica, and Lentinus tigrinus, were isolated sporadically.