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The isolation and assessment of the active constituents in polar and non-polar crude extracts of <i>Saussurea costus</i> roots as antifungal agents, against <i>Candida albicans</i> and non-<i>C</i>. <i>albicans</i> (NAC) species, was the aim of this current investigation. The SEM “Scanning electron microscopy” imaging provided potential action modes of <i>n</i>-hexane extract (<i>nh</i>hE) toward <i>Candida</i> spp., whereas the TLC-DB “Thin layer chromatography-direct bioautography” was employed for detecting the anticandidal compounds. <i>nh</i>hE had the greatest biocidal activity against all strains and clinical isolates of <i>Candida</i> spp. with maximum zones of inhibition. SEM revealed the occurrence of irregular, dense inclusions of <i>C. albicans</i> cell walls after treatment with <i>nh</i>hE for 12 h. Complete morphological distortions with lysed membranes and deterioration signs appeared in most treated cells of <i>C. parapsilosis</i>. The most effectual compound with anticandidal activity was isolated using TLC-BD and identified as sesquiterpene by GC/MS analysis. The infra-red analysis revealed the presence of lactone ring stretching vibrations at 1766.72 cm⁻¹. The anticandidal activity of <i>nh</i>hE of <i>S. costus</i> roots was confirmed from the results, and the treated cotton fabrics with <i>nh</i>hE of <i>S. costus</i> possessed observable activity against <i>C. albicans</i>. Data could recommend the practical usage of <i>S. costus</i> extracts, particularly <i>nh</i>hE, as influential natural bioactive sources for combating pathogenic <i>Candida</i> spp.