Apiognomonia hystrix is an ascomycetous fungus within Diaporthales that is found on maples and to a lesser extent on other hardwood trees in Europe, Northern America and Asia. To date, varying opinions on the species’ status as a cause of plant diseases have been expressed. In this study, we present the results of analyses conducted from 2012–2017 at forest sites in Poland on the occurrence of A. hystrix on Acer pseudoplatanus and Fraxinus excelsior and the pathogenicity of this fungus towards both tree species. For the sycamore leaves, A. hystrix conidiomata were detected in connection with 19.2% of galls caused by Dasineura vitrina, 20.4% of galls caused by Drisina glutinosa and 67.9% of extensive vein-associated necroses. The A. hystrix colonization of galls caused by both midge species resulted in statistically significantly larger necroses. On European ash leaves, conidiomata of A. hystrix occurred in connection with 0.8% of Dasineura fraxinea galls. Perithecia of A. hystrix were detected on overwintered leaf petioles in 8.1% of A. pseudoplatanus and 1.2% of F. excelsior samples. Twelve representative cultures were characterized molecularly by barcoding three marker genes (ITS, ACT, CAL). Results of phylogenetic analyses indicate that A. hystrix isolates are genetically variable, and three lineages are distinguishable. Eight isolates, including four originating from sycamore and four from European ash, were used to determine A. hystrix pathogenicity. Among the 48 A. pseudoplatanus petioles inoculated with A. hystrix, 41 developed necrotic lesions after 8 weeks, with the average necrosis length caused by particular isolates ranging from 14.5 to 67.2 mm. None of the 48 inoculated F. excelsior petioles developed necrotic lesions. Finally, selected aspects of A. hystrix morphology on natural substrates and in vitro are discussed in this paper, as well as the species’ potential to cause disease symptoms.