Costus speciosus is a unique Indian ornamental plant species in the genus Costus that has used in traditional medicine where exhibited high importance to multi-purpose pharmacological applications. Essential oil was first obtained from Costus speciosus dried roots, ethanolic extract containing diosgenine was isolated and epoxidized by using m-chloroperbenzoic acid to yield diosgenine epoxide, then analyzed them by using IR, NMR and GC-MS methods. Antimicrobial activities of the essential oil, organic extracts of the dried roots of Costus speciosus were scored on the basis of the change in the general shape of the fungal spores SEM analysis. The results showed that in vitro treatment of fungi by the essential oil led to hyphae disruptions and changes in the fungal spore shapes. The studies on the antimicrobial activity showed high growth inhibition for Microsporum gypseum, M. canis, Candida albicans, Pseudomonas aeruginosa and Staphylococcus aureus when challenged with concentrated oil extract. Methanol and ethanol extracts of the oil exhibited an inhibitory effect on the fungi, yeast and bacteria, while the constituent diosgenin epoxide ingredient in the oil showed higher in vitro growth inhibitory activity than diosgenin against M. gypseum, C. albicans and C. tropicalis.