Entomopathogenic fungi (EPFs) are a significant group of insect pathogens that are used as microbial insecticides with distinct physiological mechanisms of pathogenicity. During our survey in the middle of March 2020 to early October 2022 on natural EPF as a biological control agent of the longhorn beetle (LHB) Osphranteria coerulescens, new isolates of EPFs were found on LHB larvae in apple orchards of Dargaz, Razavi Khorasan province, Iran. The nBLAST results of this these indigenous isolate indicated the highest similarity to Cladosporium sp. (Ascomycota: Davidiellaceae) with ON307222.1 access number and for EF-1α indicated the highest similarity to Cladosporium sp. with MH724951.1 access number. To assess the virulence of these indigenous isolates of EPF, a series of tests were conducted on the LHB larvae stages. Conidial concentrations 104, 105, 106, 107, and 108 (conidia/ml− 1) caused (10.21 ± 2.1), (23.44 ± 2.4), (28.72 ± 2.6), (33.23 ± 3.1) and (34.02 ± 2.8) % of mortality in LHB, respectively and there was no significant difference between 108 and 107 (conidia/ml−1) concentrations. The mortality results by the time showed that the process did not start until 3 days after treatment, and after that, it sped up. This research gathers new information about cryptic inhabiting larvae which shows considerable potential for this Cladosporium species to be implemented within the microbial control program of the LHB. This research is a new report isolating the pathogenicity of Cladosporium sp. on LHB. These fungal isolates have considerable potential for the microbiological control of the LHB.

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