Common Sweet Flag or Acorus calamus is a medicinal plant from the Acoraceae family. Tamil name is Vashambu. It is a tall perennial wetland monocot with scented leaves and rhizomes which have been used in medicinal field. In vitro studies indicate that even those plants / plant parts / extracts that have been already evaluated for their antimicrobial efficacy have to be re-evaluated adopting these more precise and advanced assay methods. Present study was Isolation and maintaining the standard ATCC strains and clinical fungal samples. Preparation of lyophilized seitz aqueous and ethanolic extract from Acorus calamus rhizome. Find the antifungal activity of rhizome extraction of Acorus calamus using micro dilution method (M 38-P) with the RPMI-1640 medium. Find the Minimum Inhibitory Concentration (MIC) of rhizome of Acorus calamus for standard strains and clinical isolations of Candida albicans, Microsporum gypsum, Trichophyton rubrum and T. Mentagrophytes. Compare the Minimum Fungicidal Concentration of rhizome of Acorus calamus with few standard antifungal drugs such as Amphotericin B, Itraconazole and Fluconazole. The results of the present study was lyophilized ethanolic extract of Acorcus calamus rhizome also exhibited fungicidal on both standard and clinical strains showing MIC value of 12.5 µg/ml for ATCC and 1.56 µg/ml for clinical. The efficacy of the lyophilized extract can be considered highly remarkable as the MIC value of standard medicines Amphotericin B, Itraconazole and Fluconazole on Candida albicans and Trichophyton mentegrophytes and Trichophyton rubrum of Clinical strains and as well as ATCC strains.