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• Reports long term clonal fidelity validation of M. acuminata. • Determination of quercetin, p -coumaric acid and eugenol within various tissues using HPTLC. • Assessment of anti- bacterial activity of various plant parts against prominent strains. • Validation of genetic conformity using SCoT and CBDP-gene targeted markers. • Model approach for other terrestrial orchids. Malaxis acuminata D. Don [ = Crepidium acuminatum (D. Don) Szlach.] is one of the prized medicinal orchids with large-scale application in various nutraceutical and herbal preparations. However, due to paucity in the availability of plant materials, its usage is often restricted. In order to ensure continuous supply of raw material for the pharmaceutical and herbal industries, plant tissue culture plays a crucial role. In the present research, transverse thin cell layer (t -TCL) derived in vitro cultures of M. acuminata were initiated and maintained in Murashige and Skoog (MS) medium supplemented with 1.5 mg/l meta -topolin (m T), 5.0 mg/l chitosan, 1.5 mg/l indole-3- butyric acid (IBA) and 5.0 mg/l phloroglucinol (PG) for three years time period under culture room conditions by regular intermittent sub-culturing. However, the success of in vitro propagated plant bio-resources are often adversely impacted due to the occurrence of clonal variations which is considered as a serious limitation for the commercial utilization. In order to ascertain the clonal fidelity of the long term - propagated in vitro lines of M. acuminata , genetic homogeneity amongst the regenerated plantlets was accessed using two advanced gene targeted molecular markers namely start codon targeted (SCoT) and CAAT box derived polymorphism (CBDP). The plants were found to exhibit 93.34% clonal fidelity in comparison to their mother plant. Keeping in consideration the therapeutic attributes of M. acuminata , the phytochemical makeup of the wild and in vitro - derived plant parts was compared using high performance thin layer chromatography (HPTLC) technique. Furthermore, M. acuminata is considered to be one the most precious medicinal orchids having rich reserves of biomolecules which can be used against various pathogenic bacterial strains. The antibacterial activities of various tissue extracts of M. acuminata were also evaluated against the opportunistic bacterial strains viz. Escherichia coli (MTCC43), Salmonella typhimurium (MTCC733), Staphylococcus aureus (MTCC96) and Bacillus subtilis (MTCC121). The results of antibacterial assays demonstrated the therapeutic efficacy of the in vitro - derived plant parts at identical magnitude with that of their wild counterparts. The present research provides a holistic insight into the clonal stability of the long term - propagated in vitro plants of M. acuminata with promising antibacterial activity. This study can also serve as a model approach for the maintenance of genetically stable in vitro lines of therapeutically important endangered orchid species over a period of time. [ABSTRACT FROM AUTHOR]