The acreage of English walnut (Juglans regia L.) is constantly expanding in Hungary, due to the favorable climatic conditions and economic importance. Last years, serious damage was reported from several orchards with high percentage of rotted, moldy kernels. The aim of this research was to identify the pathogens at different growth stages. Fungi were cultured from the spotty, shriveled and rotted kernels, and monosporic isolates were identified based on morphological characters and molecular markers (ITS region and tef1 locus sequences). Botryosphaeria dothidea and Diaporthe eres were identified in high proportion from symptomatic kernels. These species were also isolated from different parts of walnut trees in different seasons. D. eres was detected in a high proportion from asymptomatic buds in March, while the presence of both species was observed in symptomatic husks with Overnight Freezing-Incubation Technique (ONFIT) in June. Their optimal growth temperature defined to be between 20–25 °C, and the growth of D. eres isolates was completely inhibited at 35 °C.