BACKGROUND: Dendrobium orchids (Dendrobium spp.) are valuable medicinal and attractive ornamental plants. Due to their limited wild resources, the size of the Dendrobium spp. population required for market demand primarily depends on artificial cultivation. However, the nutritional and therapeutic value of natural products may differ as growth conditions change. In this study, we profiled metabolites from wild and cultivated Dendrobium flexicaule (D. flexicaule) to explore the variations and interrelationships among bioactive components. RESULTS: A total of 840 annotated metabolites were discovered, 231 of which differed significantly between wild and cultivated D. flexicaule. A comparative investigation found that the types and amounts of metabolites, particularly flavonoids, lipids, amino acids and their derivatives, varied between wild and cultivated D. flexicaule. Using metabolite correlation analysis, a series of differentially abundant metabolites were found to be significantly correlated with phytohormones such as abscisic acid (ABA), salicylic acid (SA), and zeatins, indicating that plant hormones play a role in the accumulation of specific metabolites. Furthermore, many distinct metabolites were identified as key active ingredients of traditional Chinese medicines. Additionally, 78 components were discovered to be active pharmaceutical substances against various diseases, probably contributing to the diverse medical values of wild and cultivated D. flexicaule. CONCLUSIONS: Overall, comprehensively analyzed the metabolic profiles of wild and cultivated D. flexicaule in this study, serving as a theoretical and material foundation for quality control, health efficacy, and industrial development.