Abstract: This paper aims to isolate endophytic bacteria from the stems of Dendrobium officinale and explore their antibacterial activity and secondary metabolites. In this study, the traditional symbiotic bacteria separation method was improved, and 16S rRNA gene sequencing technology was used for identification. The antibacterial experiments of symbiotic bacteria and their fermentation crude extracts were carried out by confrontation method and filter paper diffusion method. Finally, the secondary metabolites of strains with excellent antibacterial activity were isolated and purified by different chromatographic techniques. As a results showed, seven strains of endophytic bacteria were newly reported from D. officinale by using the improved method. The most similar species were Pantoea cypripedii, Bacillus stercoris, Microbacterium hatanonis, Pseudomonas rhodesiae, Luteibacter jiangsuensis, Janibacter melonis and Pantoea agglomerans. Among them, M. hatanonis and L. jiangsuensis were first discovered in orchids. Bacillus velezensis J-15 has the most significant antibacterial effect in endophytic bacteria. Therefore, three compounds were isolated and purified from the antibacterial site of the strain, and identified as uracil (1), 3, 4-dihydro-4-hydroxynaphthalene-2-carboxylic acid (2) and epichlicin (3). Compounds 2 and 3 were obtained from the fermentation broth of the strain for the first time. The antibacterial activity screening results showed that epichlicin had antibacterial activity against Candida albicans.