The stems of Dendrobium chrysotoxum (DC) are commonly used as health-promoting foods due to their excellent biological activities. However, the stems of D. thyrsiflorum (DT) are often used to meet the scarcity of DC in production because of their highly similar morphology. However, the related metabolomic and bioactive information on the stems of DC and DT are largely deficient. Here, secondary metabolites of DC and DT stems were identified using an ultra-performance liquid chromatography-electrospray ionization-mass spectrometry, and their health-promoting functions were evaluated using several in vitro arrays. A total of 490 metabolites were identified in two stems, and 274 were significantly different. We screened out 10 key metabolites to discriminate the two species, and 36 metabolites were determined as health-promoting constituents. In summary, DT stems with higher extract yield, higher total phenolics and flavonoids, and stronger in vitro antioxidant activities demonstrated considerable potential in food and health fields.