The ethyl acetate fraction of the dried aerial parts of Senecio glaucus L. exhibited significant antimicrobial activity against some of selected bacteria and fungi. Also, it showed potent cytotoxicity against PANC-1 cancer cell lines under glucose deficient medium. The ethyl acetate fraction was subjected to different chromatographic techniques for isolation of the bioactive compounds. A new benzofuran glucoside; 2,3-dihydro-3b-hydroxyeuparin 3-Oglucopyranoside (1) was isolated. Additionally, two known flavonoid compounds isorhamentin 3-O-b-D-glucoside (2), and isorhamentin 3-O-b-D-rutinoside (3) were first identified in S. glaucus. Compound 1 exhibited potent antimicrobial activities against two Gram-positive bacteria, one Gram-negative bacteria, and two fungi. Also, it displayed potent cytotoxic activity against PANC-1 cancer cell lines under glucose deficient medium (IC50 7.5 lM). However, the isolated flavonoid glycosides (2 & 3) showed moderate antimicrobial activities against two Gram-positive bacteria, two Gram-negative bacteria, four fungi, and did not show any cytotoxic activity against PANC-1.