Orchids are one of the immense ecological and evolutionary momentous plants and have effectively colonized almost every habitat on the earth. The seeds of Pholidota imbricata W. J. Hook. were aseptically grown on four nutrient media namely KC, MS, PM, MVW and these successfully germinated on all of the tested media. The highest percentage of seed germination (80.00%) was achieved on MS medium and greenish protocorms were obtained. During phytochemical screening, secondary metabolites i.e. alkaloids and ten other compounds namely, steroids, flavonoids, glycosides, phlobatannins, terpenoids, quinine, coumarin, anthroquinones, saponins, and tannin were recorded in both natural and in vitro developed plant parts. The comparative results of the alkaloid test revealed that in vitro developed plant parts showed better results than those of naturally grown ones. Comparative results of ten secondary metabolites tested in naturally grown plant parts were better than in vitro grown plantlets. The current investigation provides new inventions on the competency of P. imbricata and enhances the continued inquiry of medicinal orchids, in Bangladesh.