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Aims Elucidating the identity of an isolate of Aspergillus sp. obtained during searches for anti-coffee leaf rust (CLR) biocontrol agents, from healthy coffee berry samples, preliminarily verify whether it is an aflatoxin-producer, confirm its ability to grow as an endophyte in healthy coffee tissues and assess its biocontrol potential against CLR. Methods and results One, among hundreds of fungal isolates fungus were obtained from healthy coffee tissues belonged to Aspergillus (isolate COAD 3307). A combination of morphology features and molecular analyses; including four regions—internal transcribed spacer, second-largest subunit of RNA polymerase (RPB2), β-tubulin (BenA) and calmodulin (CAL)—identified COAD 3307 as Aspergillus flavus. Inoculations of healthy Coffea arabica with COAD 3307 confirmed its establishment as an endophyte in leaves, stems, and roots. Treatment of C. arabica plants by combinated applications of COAD 3307 on aerial parts and in the soil, significantly (P  > .0001) reduced CLR severity as compared to controls. Thin-layer chromatography indicated that COAD 3307 is not an aflatoxin-producing isolate. In order to confirm this result, the extract was injected into high-performance liquid chromatography system equipped with a fluorescence detector, and no evidence of aflatoxin was found. Conclusions COAD 3307 is an endophytic isolate of A. flavus —a species that has never been previously recorded as an endophyte of Coffea spp. It is a non-aflatoxin producing strain that has an anti-CLR effect and merits further evaluation as a biocontrol agent. [ABSTRACT FROM AUTHOR]