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Previous studies carried out in our laboratory described the antimicrobial activity of the whole hexanic extract (HE) of Achyrocline satureioides (Lam.) DC against Paenibacillus larvae, the causal agent of American Foulbrood (AFB) a disease of the honey bee larvae. In this study, the HE was partitioned into five main fractions by chromatographic techniques leading to the isolation of four known compounds: two prenylated phloroglucinol α-pyrones (1 and 3), 5,7-dihydroxy-3,8-dimethoxyflavone (gnaphaliin A) (2), and 23-methyl-6-O-demethylauricepyrone (4). Isolated compounds were further analyzed towards structural elucidation using 1 H RMN and 13 C RMN spectroscopic techniques. For the first time, the antimicrobial activity of the isolated compounds was evaluated against P. larvae strains by broth microdilution method and compared with that of the whole HE. Compounds 1-4 displayed minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranging between 0.07 and 62.5 μg/mL and 0.26 and 12.5 μg/mL, respectively. The lowest MIC and MBC values were obtained with compounds 3 and 4, respectively. The antimicrobial activity of each single compound and the combination of them showed that the presence of all compounds is needed for the antimicrobial efficacy of whole HE., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)