The aim of this study was the isolation and structural elucidation of steroidal glycosides from the underground parts of ramson Allium ursinum L. The structures of the isolated compounds were established based upon chromatographic methods and 1D- and 2D-NMR, MS and IR analyses. The mixture of two steroidal saponins: (25R)-spirost-5-en-3b-ol tetrasaccharide and (25R)-spirost-5, 25(27)-dien-3b-ol tetrasaccharide, along with a 3-hydroxypregna-5,16-dien-20-one glycoside were identified. The results of in vitro cytotoxic activity of the mixture of spirostanol saponins against cell lines melanoma B16 and sarcoma XC and human fibroblasts HSF are also reported. The spirostanol saponins mixture was investigated to determine its in vitro antimicrobal activity against Trichophyton mentagrophytes and Microsporum canis.