Three antimicrobial sphingolipids were separated by bioassay-guided isolation from the chloroform fraction of the crude methanol extract of cucumber (Cucumis sativus L.) stems and identified as (2S,3S,4R,10E)-2-[(2'R)-2-hydroxytetra-cosanoylamino]-1,3,4-octadecanetriol-10-ene (1), 1-O-β-D-glucopyranosyl(2S,3S,4R,10E)-2-[(2'R)-2-hydroxy-tetracosanoylamino]-1,3,4-octadecanetriol-10-ene (2) and soya-cerebroside I (3) by their physicochemical properties and spectroscopic analysis. They were evaluated to show antifungal and antibacterial activity on test microorganisms including four fungal and three bacterial species. Among them, compound 1, a relatively low polarity aglycone, exhibited stronger antimicrobial activity than its corresponding glycoside 2. The results indicated that sphingolipids could be the main antimicrobial compounds in the crude methanol extract of cucumber stems.