Juglans regia L. (English walnut) trees with cankers and dieback symptoms were observed in two regions in the Czech Republic. Isolations were made from diseased branches. In total, 138 fungal isolates representing 10 fungal species were obtained from wood samples and identified based on morphological characteristics and molecular methods: Cadophora novi-eboraci, Cadophora spadicis, Cryptovalsa ampelina, Diaporthe eres, Diplodia seriata, Dothiorella omnivora, Eutypa lata, Eutypella sp., Peroneutypa scoparia, and Phaeoacremonium sicilianum. Pathogenicity tests conducted under field conditions with all species using the mycelium-plug method indicated that Eutypa lata and Cadophora spp. were highly virulent to woody stems of walnut. This is the first study to detect and identify fungal trunk pathogens associated with diseased walnut trees in Europe.