This work aims to produce, identify, and characterize the bioactive surfactant from Enterococcus faecium NM113, previously isolated as probiotic from the feces of breast-fed infants according to its immunomodulatory properties. The yield of the biosurfactant (BS) production was as 0.2 g L^-1.   Functional and structural characterizations of the produced biosurfactant were achieved by TLC followed by post chromatographic detection. Silica plate or replica plate was sprayed with anthrone produced gives a yellow spot indicated the presence of sugar moieties. Lipopeptide residues were detected while stained with Ninhydrin reagent. According to FTIR spectrum obtained, the extracted BS is composed of carbohydrates and lipid. The hydrophobic chain of the BS is composed of lipid and hydrophilic part is mainly composed of sugar. Furthermore, Identification of BS features was done by ¹H NMR and ¹³C NMR and confirmed as glycolipid. The evaluation of the antimicrobial activity of BS produced from E. faecium NM113 was done against five pathogens. The biosurfactants have a great opportunity to be novel agents to compete the antimicrobial-resistance microorganisms.