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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 <i>Neopestalotiopsis clavispora</i> through morphological observation, molecular identification, and multi-gene (ITS, <i>TEF1α</i>, and <i>TUB2</i>) 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 <i>N. clavispora</i> being responsible for apple leaf spots in China, and the disease has been named ‘apple Neopestalotiopsis leaf spot’. Additionally, <i>N. clavispora</i> was found to infect crabapple, sweet cherry, grape, peach, and pear under laboratory conditions, indicating that these fruit trees may be potential hosts for <i>N. clavispora</i> 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 <i>N. clavispora</i>. Among them, those based on pylocyanonitrile, propiconazole, pyraclostrobin, tebuconazole, diphenoxazole, and osthole showed higher toxicity to <i>N. clavispora</i>, 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 <i>N. clavispora</i> causing apple leaf spot and are crucial for developing effective management strategies.