Bacterial volatile organic compounds (VOCs) have been considered as sensitive and specific biomarkers for bacterial detection in human specimens and culture media. The possibility of using VOCs markers as one of the largest groups of bacterial metabolites would open a new frontier for developing more efficient techniques in the diagnosis of bacterial infections. The aims of this research were analysis of the bioactive chemical products and evaluation of antibacterial and antifungal activity. Bioactives (chemical compounds often referred to as secondary metabolites) were analyzed using gas chromatography-mass spectroscopy (GC-MS) techniques, then the in vitro antibacterial and antifungal activity of the methanolic extract was evaluated. Thirty five bioactive compounds were identified in the methanolic extract of Staphylococcus aureus. GC-MS analysis of Staphylococcus aureus revealed the existence of the: Hexanoic acid, 2-methyl, 12,15-Octadecadiynoic acid, methyl ester, 1-Aminononadecane,N-trifluoroacetyl-, N-[3-[N-Aziridyl]propylidene]hexylamine, N-(2,5-Dicyano-3,4-dihydro-2H-pyrrol-2-yl)-acetamide, 3-Cyclohex-3-enyl-propionic acid, 1-Methyl-4-[nitromethyl]-4-piperidinol, 3-Azonia-5-hexyn-1-ol, N,N-dimethyl-O-acetyl-,bromide, 1-Hexadecanol-2-methyl-, 1-Propyl-3,6-diazahomoadamantan, 9-Borabicyclo[3.3.1]nonane, 9-mercapto-, Benzyl methyl ketone, L-Aspartic acid,N-glycyl-, Aminoacetamide, N-methyl-N-[4-(1-pyrrolidinyl)-2-butynyl]-, Tertbutyloxyformamide, N-methyl-N-[4-(1-pyrrolidinyl)-2-butyn, 5,7-Dodecadiyn-1,12-diol, Deoxyspergualin, D-Streptamine, O-6-deoxy-?-D-glucopyranosyl-(1-4), dl-Citrulline, N-[3-Diethylaminopropyl]-4-oxo-1,2,3,4,5,6,7,8-octahydroqui, N-Propionyl-D-glucoseamine, Cystine, 3,4-Dihydrocoumarin,6-fluoro-4,4-dimethyl-, 4-(2,5-Dihydro-3-methoxyphenyl)butylamine, 3-methoxy-2-(1-methylethyl)-5-(2-methylpropyl)pyrazine, Uric acid, Thiocyanic acid 4-methoxy-2,6-dimethyl-3-pyridyl ester, 12-Dimethylamino-10-oxododecanoic acid, Glycyl-D-asparagine, Actinomycin C2, 12-Octadecenoic acid, methyl ester, 6-Octadecenoic acid, methyl ester,(z)-, 2,5-Piperazinedione, 3,6-bis(2-methylpropyl)-, l-Leucyl-d-leucine and Methyl 12-hydroxy-9-octadecenoate. Gramineae poaceae was very highly active (6.71±0.13) mm. The results of anti-fungal activity produced by Staphylococcus aureus showed that the volatile compounds were highly effective to suppress the growth of Aspergillus terreus. Staphylococcus aureus produce many important secondary metabolites with high biological activities. Based on the significance of employing bioactive compounds in pharmacy to produce drugs for the treatment of many diseases, the purification of compounds produced by Staphylococcus aureus can be useful.