Alpinia galanga, a medicinally important rhizomatous plant, is valued for its bioactive compounds and essential oils, which exhibit diverse pharmacological activities. The present study evaluated the in vitro culture establishment, phytochemical composition, and antioxidant potential of A. galanga. Callus induction from explants was strongly influenced by growth regulator treatments. Treatment with 2,4-D at 2 mg L⁻¹ yielded the highest embryogenic callus induction (48.63%), while higher concentrations were inhibitory. The combination of 2 mg L⁻¹ 2,4-D with 0.3 mg L⁻¹ TDZ significantly enhanced induction (76.84%), demonstrating synergistic effects. Shoot regeneration from embryogenic cell suspensions was maximized with 2 mg L⁻¹ BA alone, achieving 75.24% regeneration and 11.46 shoots per callus, whereas BA–kinetin combinations reduced efficiency. GC-MS analysis of conventionally propagated and callus-derived leaf and rhizome oils identified 45 volatile compounds, predominantly monoterpenes, sesquiterpenes, and phenylpropanoids. Major constituents such as eucalyptol, β-pinene, and camphor were retained, while guaiol and fenchyl acetate showed increased accumulation in callus-derived oils. Callus induction also enhanced total phenolic (TPC: 84.38 ± 1.03 mg GAE/g) and flavonoid contents (TFC: 75.33 ± 1.17 mg QE/g) compared to conventional propagation. Antioxidant activity assessed via DPPH assay revealed strong radical scavenging potential, with callus-induced rhizome oil exhibiting the lowest IC₅₀ (14 µg/ml), surpassing ascorbic acid (18 µg/ml). Overall, in vitro culture effectively preserves phytochemical profiles while enhancing bioactive compound accumulation and antioxidant capacity in A. galanga, highlighting its potential for sustainable production of natural antioxidants.