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• 12 isoflavonoid compounds identified out of 38 detected of EERBP by HPLC-ESI/MS. • Inhibition (81%) on mycelial growth of C. musae by EERBP (1.6 g GAE L−1). • Two fractions of EERBP detected and isolated by TLC-bioautography method. • An additive effect observed through the biological activity of these 2 fractions. This study set out to highlight the in vitro and in vivo antifungal activity of an Ethanolic Extract of Red Brazilian Propolis (EERBP) and identify bioactive fractions effective against Colletotrichum musae. Active fractions were detected by the thin-layer chromatography-bioautography method and characterised by HPLC-MS n. The in vitro results showed that EERBP had strong antifungal properties against C. musae (81 ± 1% inhibition at 1.6 g GAE L−1). Medicarpin, (3 S)-vestitol and (3 S)-neovestitol were the main compounds identified in the EERBP extract (45% of all detected peaks). Two isolated fractions displayed inhibition percentages of 35 ± 4 and 42 ± 1%, respectively, on C. musae mycelial growth compared to the EERBP extract. The biological activity of the two fractions displayed an additive effect. A further in vivo investigation revealed that EERBP is a potential natural alternative for controlling banana crown rot. [ABSTRACT FROM AUTHOR]