Antimicrobial activity of solvent extracts and flavonoids of Calotropis procera growing wild in Saudi Arabia was evaluated using the agar well-diffusion method. A bioassay-guided fractionation of the crude flavonoid fraction (Cf3) of MeOH extract which showed the highest antimicrobial activity led to the isolation of four flavonoid glycosides as the bioactive constituents. Structure of compounds have been elucidated using physical and spectroscopic methods including (UV, IR, (1)H, (13)C-NMR, DEPT, 2D (1)H-(1)H COSY, HSQC, HMBC and NOESY). Compounds were found to be the 3-O-rutinosides of quercetin, kaempferol and isorhamnetin, besides the flavonoid 5-hydroxy-3,7-dimethoxyflavone-4'-O-β-glucopyranoside. Most of the isolated extracts showed antimicrobial activity against the test microorganisms, where the crude flavonoid fraction was the most active, diameter of inhibition zones ranged between 15.5 and 28.5 mm against the tested bacterial strains, while reached 30 mm against the fungal Candida albicans. The minimal inhibitory concentrations varied from 0.04 to 0.32 mg/ml against all of the tested microorganisms in case of the crude flavonoid fraction. Quercetin-3-O-rutinoside showed superior activity over the remainder flavonoids. The Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) were more susceptible than the Gram-negative (Pseudomonas aeruginosa and Salmonella enteritidis) and the yeast species were more susceptible than the filamentous fungi. The study recommend the use of such natural products as antimicrobial biorationals.