The antibacterial effect of the crude hot Chloroform extracts and purified fractions of Spirogyra sp. (Green algae) against multidrug resistant human pathogen were isolated from burn and bound. The test bacterial strains were Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris and Escherichia coli. Hot Chloroform extracts (128 and 256 mg/mL) of Spirogyra sp. inhibited growth of all the test organisms. Primary detection of active compounds showed that (Spirogyra sp.) containing Terpenoid, Flavonoids ,Phenols Saponins and Alkaloids. Gas Chromatography-Mass Spectrometry was used to know the compounds which responsible of antibacterial activity and they wereNonadecane (44.5%) and Eicosane(19.2%) are alkane hydrocarbon Alkanes .While, Pentadecane represented(10.2%).Octadecane(8.3%),Tetradecanedihydroxyl (4.2%) Hexadecane 2-hydroxyl(2.1%) and Hexadecane(1.3%) from the crud hot extract of Spirogyra sp.These findings suggest the possibility of using the Spirogyra sp. as a novel source of natural antimicrobial agents in pharmaceutical industries.