Swertia corymbosa (Griseb.) Wight ex C. B. Clarke, a valuable medicinal plant, has been investigated for its regeneration potential using nodal explants. Out of a range of concentrations of cytokinins [6-benzyl adenine (BA), 6-furfurylaminopurine (Kn), 2-isopentenyl adenine (2iP), thidiazuron (TDZ), and zeatin (Z)] used as supplements with MS, BA at 4.40 μM concentration proved best for multiple shoot induction yielding 26.50 ± 0.26 shoots after 12 weeks of culture. Addition of low concentration of NAA (1.3 μM) in MS medium supplemented with the cytokinin BA (4.40 μM) favoured shoot multiplication. A mean number of 35.78 ± 0.81 shoots were produced per explant. Additive effect of BA (4.40 μM) in combination with Kn (4.64 μM) produced highest number of shoots (83.20 ± 4.29). Addition of GA₃ (1.4 μM) to the above medium not only favored shoot elongation but also enhanced the number of shoots (113.98 ± 3.80). The microshoots were rooted successfully on half-strength MS medium supplemented with 9.8 μM of IBA. The plantlets were successfully transferred to hardening medium containing vermiculite with 87 % survival rate. Screening of the antibacterial, antioxidant activity and estimation of total phenolic and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolic and flavonoid content than in vitro propagated plants. The content of secondary metabolites in wild-grown plants and in vitro propagated plants was determined by HPLC coupled with ESI-MS and the presence of loganic acid, swertiamarin, sweroside, gentiopicroside, isovitexin, amoroswertin, amarogentin, gentiacaulein, decussatin, and swertianin in the samples were confirmed. Gentiopicroside (40.726 mg/g) and swertianin (29.598 mg/g) were found to be the major compounds which may be responsible for the antimicrobial and antioxidant activities. The results of the present study confirmed the therapeutic potency of S. corymbosa used in the traditional medicine; in addition, the protocol for in vitro production developed in the present study could be applied for mass multiplication and for the conservation of germplasm.