Objectives To determine the secondary metabolite content, antioxidant and phenylalanine ammonia-lyase (PAL) activity as well as essential oil composition of in vitro cultures and field grown rhizomes of Zingiber montanum. Results Methyl jasmonate-treated cell cultures showed the highest total phenolic content and steroid content of 22.23 mg gallic acid equivalent/g dry weight (DW) and 41.67 mg/g DW, respectively. Callus cultures exhibited the highest tannin content (39.53 mg tannic acid equivalent/g DW) and strongest antioxidant activity (91.05% inhibition of 2,2-Diphenyl-1-picrylhydrazyl or DPPH). The highest saponin (81.76 mg/g DW) and alkaloid (113.97 mg/g DW) contents were obtained in in vitro microrhizomes induced on Murashige and Skoog (MS) medium supplemented with 6% sucrose and 5 mg/l 6-Benzylaminopurine (BAP), and MS medium supplemented with 7% sucrose, respectively. The essential oil content varied in cell cultures and microrhizomes and mainly consisted of fatty acid esters, which are precursors of many secondary metabolites. Trace amounts of terpinen-4-ol (0.21 and 0.27 mg/g) and zerumbone (0.0107 mg/g) were also detected in the in vitro microrhizomes. Conclusion The results obtained indicate the potential of in vitro cultures of Z. montanum for the production of secondary metabolites.