[Objective] To isolate and identify endophytic bacteria from the roots of Paeonia suffruticosa and evaluate antifungal activity of secondary metabolites of antagonistic strains in vitro. [Methods] The endophytic bacteria were screened against Botrytis paeoniae, Gloeosporium sp., Altenaria sp., Phyllosticta commonsii through the method of dual culture. The strains were indentified based on characteristics in morphology, physiology, biochemistry and phylogenetic analysis of 16S rRNA gene sequences. The lipopeptide biosynthetic genes fragments of antagonistic strains were amplified by polymerase chain reaction. The crude lipopeptides of strains were obtained through acid precipitate method. The inhibition ability of lipopeptides produced by antagonistic strains against pathogenic fungi was evaluated in vitro. [Results] The 62 endophytic bacterial isolates were isolated from the roots of Paeonia suffruticosa. The isolates Md31 and Md33 showed inhibitory activity against four pathogens. Md31 and Md33 were identified as Bacillus amyloliquefaciens. The genes fragments of bmyB, fenD, ituC, srfAA and srfAB were obtained in genomic DNA of the two strains. The crude lipopeptides prouduced by Md31 and Md33 were isolated from culture supernatants and exhibited inhibitory activity on mycelium growth of peony pathogens. [Conclusion] Two antagonistic B. amyloliquefaciens strains Md31 and Md33 were obtained, and their crude lipopeptides showed inhibitory activity against four peony pathogens in vitro, which will been provided a basis for the further development and application of peony endophytes.