Leaf spot, a major apple disease, manifests in diverse symptoms. In this study, the pathogen was isolated from diseased ‘Yanfu 3’ apple leaves in Yantai, Shandong Province, and identified as Neopestalotiopsis clavispora through morphological observation, molecular identification, and multi-gene (ITS, TEF1α, and TUB2) phylogenetic analysis. Three isolates (YTNK01, YTNK02, and YTNK03) were selected for pathogenicity tests to verify Koch’s postulates. To our knowledge, this is the first report of N. clavispora being responsible for apple leaf spots in China, and the disease has been named ‘apple Neopestalotiopsis leaf spot’. Additionally, N. clavispora was found to infect crabapple, sweet cherry, grape, peach, and pear under laboratory conditions, indicating that these fruit trees may be potential hosts for N. clavispora in the field. The in vitro toxicity of ten fungicides to the pathogen was assessed using the mycelial growth rate method. All ten fungicides were effective in inhibiting the growth of N. clavispora. Among them, those based on pylocyanonitrile, propiconazole, pyraclostrobin, tebuconazole, diphenoxazole, and osthole showed higher toxicity to N. clavispora, with EC₅₀ values of 0.11, 0.41, 0.47, 1.32, 1.85, and 3.82 µg/mL, respectively. These fungicides could be used as alternatives to prevent this disease in production. Overall, these findings provide valuable insights into the characteristics of N. clavispora causing apple leaf spot and are crucial for developing effective management strategies.