Curcuma larsenii Maknoi & Jenjitikul is a member of the Zingiberaceae family, possessing significant pharmacological potential, although it has become endangered through the abuse of resources. This research article delineates the findings of the in vitro propagation, transplantation, and phytochemical profiles of C. larsenii, a rare plant species in Thailand. Microshoots measuring 1 cm in length were used as explants for the induction of shoots and roots in both solid and liquid Murashige and Skoog medium, incorporating various concentrations of cytokinins (6-benzylamino-purine (BA), 6-furfurylaminopurine (kinetin), thidiazuron (TDZ)) and auxins (1-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA)) over a duration of 8 weeks. This study assessed the total phenolic content, total flavonoid content, and antioxidant activity via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays and conducted high-performance liquid chromatography (HPLC) analysis. The highest number of shoots was recorded in solid and liquid media containing MS medium enriched with 2 mg/L BA and 0.5 mg/L NAA, as well as 2 mg/L BA and 0.5 mg/L IAA, yielding 5.40 and 8.80 shoots/explant, respectively. The biggest roots/explant induction of 9.20 was attained using the liquid MS medium supplemented with 4 mg/L BA and 0.5 mg/L IAA. The highest survival rate (100%) was recorded when tissue culture plantlets were transplanted into a mixture of sand and soil (1:1). In vitro-cultivated plants exhibited superior total phenolic content relative to wild plants. Leaf extracts of C. larsenii exhibited markedly superior antioxidant activity compared to other plant organs from both in vitro and wild specimens. C. larsenii wild plants and in vitro plants generated phenolic acids and flavonoids and exhibited antioxidant activity, demonstrating a biotechnological alternative for the acquisition of bioactive substances.