Saussurea costus (Sc) plant belongs to the Asteraceae family. It contains various bioactive compounds used traditionally in treating multiple diseases. This study intends to extract and evaluate the active ingredients of Sc roots (Scrs) and determine their antibacterial activities. S. costus roots were purchased from the herbal market and the chemical compositions, including protein, lipid, carbohydrates, moisture, and ash, were determined according to AOAC procedures 2016 with some modifications. HPLC and amino acids analyzer were used to determine the phenolic compounds and amino acid composition of S. costus roots extract (ScrsE). The antibacterial activities of S. costus roots extract were done according to the Kirby-Bauer disc diffusion method. The S. costus roots were revealed a fusiform (or) conical and tapering appearance, collapsing in the centre with longitudinal wrinkles revealing 11-19 cm and 1-2.5 cm for long and wide, respectively. The extraction yield was 9.52% and the weight of the concentrated crude extract was 10 g. According to Proximate analysis, the S. costus roots extract showed 3.88% and 20.12% moisture and ash contents, respectively. The compositions of carbohydrates, protein, and crude fat were 75.25%, 2.51%, and 1.85%, respectively. HPLC analysis showed that the S. costus roots Extract contained two phenolic acids and two flavonoids. Moreover, twelve amino acids were determined for the first time in the S. costus roots extract. Various antibacterial activities were seen with maximum inhibition zone for Streptococcus pyogenes & E.coli; and minimum for Bacillus subtilis & Pseudomonas aeruginosa. In conclusion, this study investigated various contents of S. costus roots extract including amino acids, carbohydrates, protein, and crude fat, in addition to its antibacterial activities. The authors recommend more future studies to investigate the therapeutic activities of S. costus roots extract.