Saussurea costus had a wide range of antimicrobial activities which used as alternative for synthetic preservatives that threaten human health. This study aimed to identify the bioactive compounds in S. costus extract (SCE) and to evaluate its antimicrobial activity against some pathogenic microorganisms. HPLC and GC-MS were used to quantify the bioactive compounds in SCE. The results indicated that ethanol and ethyl acetate extracts had the highest levels of polyphenols followed by n-butanol, and then n-hexane extracts. The main phenolic compounds are Naringenin, Chlorogenic acid, Ferulic acid, Ellagic acid, Gallic acid and coffeic acid followed by taxifolin, catechin, syringic acid, methyl gallate, vanillin, kaempferol, cinnamic acid and rutin. GC-MS results showed 14 compounds in S. costus extract. The antibacterial activity of S. costus ethanol extract increased by increasing the concentration of extract from 10 µl to 50 µl for each wells .The inhibition zones were 13 mm and 23mm for S. typhi and Staphylococccus aureus, respectively. Gram (+ve) bacteria found to be more sensitive to SCE than Gram (-ve) bacteria. Similarly; the antifungal activity was increased by increasing the concentration of SCE the inhibition zones were 15.5 mm and 22.5 mm for P. verecossum and A. ochraceous, respectively. A. ochraceous appeared to be more sensitive towards all concentration of the extract. The minimum inhibitory concentration (MIC) of SCE for both bacteria and fungi strains ranged from 0.08 -0.3 mg/ml and 0.25 -1.17 mg/ml, respectively. The results revealed that the SCE can play an important role against the human multi-drug resistant pathogens and can alternate the antibiotics as well as chemical preservatives to control infection and food spoilage contaminants.