Dieback disease has been reported to cause significant losses in various perennial crops worldwide. In recent years, dieback symptoms have been observed in Australian persimmon orchards, leading to a decline in plant health and the death of a significant number of trees within 6 to 36 months of establishment. Mature plants and the graft union of planting material exhibit staining of internal vascular tissue. Reports of persimmon dieback indicate a diverse range of fungal species is responsible for the disease, with variance between different countries. In Australia, the aetiology and epidemiology of the causal pathogen have not been elucidated. This study aimed to investigate the cause of persimmon dieback across persimmon-growing regions in Australia. Samples exhibiting dieback symptoms from both established trees and nursery materials were collected and processed in the laboratory. To identify the causal pathogen, diseased tissues were surface-sterilised and plated onto potato dextrose agar with chloramphenicol (PDA-C), dichloran-rose bengal chloramphenicol (DRBC) agar, and nutrient agar (NA). The isolations confirmed the presence of fungal pathogens, while no bacterial isolates were recovered. Cultural characterisation revealed that the fungal colonies had a white, cottony appearance with aerial mycelium. Morphological analysis of spores revealed fusiform conidia with 3–4 septa and a thread-like apical appendage arising from the terminal cell. Based on these cultural and morphological characteristics, the pathogen closely resembled species of Neopestalotiopsis. Pathogenicity testing, including detached stem assays and glasshouse trials, confirmed Neopestalotiopsis sp. as a causal agent of persimmon dieback. In the detached stem assay, fungal isolates were inoculated onto wounded persimmon stems. After one month, symptoms developed, and re-isolation confirmed Koch’s postulates. Representative pathogenic isolates were then inoculated into glasshouse-grown persimmon trees and monitored for 12 months to assess disease progression. Both the detached stems and glasshouse-inoculated trees exhibited internal vascular staining, further confirming the pathogenicity of Neopestalotiopsis. In glasshouse-inoculated trees, re-isolations were performed at 1 cm intervals above and below the inoculation point to assess the extent and direction of disease progression. Results after 12 months showed that the pathogen could still be re-isolated up to 2 cm from the inoculation point in both directions. These findings may provide insight into pruning recommendations, suggesting that removing at least 2 cm beyond visible symptoms may help reduce the progression of disease. While Neopestalotiopsis has previously been reported to cause leaf spots in persimmons, it has never been associated with dieback disease. This study provides the first report of Neopestalotiopsis as a persimmon dieback pathogen. Species-level identification using a multi-gene PCR assay is currently underway.