BACKGROUND: Dendrobium amoenum is known for its aesthetic and medicinal values but it is threatened due to loss of wild resources. Plant tissue culture promotes wild resource protection and paves the way for secondary metabolite production. In this study, protocorms developed via in-vitro seed cultivation were used for bioactive secondary metabolite production. The objectives of this study were to evaluate total phenolic and flavonoid contents, to identify the bioactive secondary metabolites, to explore the antioxidants and cytotoxic properties of in-vitro-derived protocorms extracts of D. amoenum. METHODS: Seeds of D. amoenum were cultivated on 10% coconut water, 0.25 and 0.5 mg/L BAP supplemented full-strength and half-strength MS medium to produce protocorms for the isolation of bioactive components. A distinct yellow fraction (DAYF), light-green fraction (DALGF), green fraction (DAGF), and dark-green fraction (DADGF) were obtained from methanol extract on a methanol-based Sephadex LH-20 column. The total phenol and flavonoid contents along with the antioxidant and cytotoxic properties of the fractions were evaluated. The compounds in active DAYF were identified using a GC-MS. RESULTS: On a full-strength solid MS medium supplemented with 10% coconut water, approximately 95% of the seeds grew into protocorms, while 88.33% did so on a full-strength liquid MS medium. The DAYF had a total phenol content of 206.38 μg of GAE and a total flavonoid content of 101.88 μg of QE. Owing to these high contents, the DAYF inhibited 50% of the DPPH free radicals at a concentration of 63.73 μg/ml. Similarly, it also reduced the growth of HeLa cells by 50% at 67.03 μg/ml and U2OS cells by 50% at 207.40 μg/ml, while it was nontoxic to normal human epithelium cells. Bioactive phenolic compounds 2-methoxy-4-vinylphenol (1), 3,4-dimethoxy-phenol (2), 2-methoxy-4-(1-propenyl)-phenol (3), 2,6-dimethoxy-4-(2-propenyl)-phenol (4), 3-methoxy-1,2-benzenediol (5) were identified in the DAYF. CONCLUSION: Protocorms of D. amoenum could serve as sources of bioactive secondary metabolites highlighting their potential in alternative medicine.