The strain Bacillus amyloliquefaciens S5I4 (B. amyloliquefaciens) isolated from Idfou Temple, Egypt, was identified by 16S rRNA and has accession number AB813716. B. amyloliquefaciens S5I4 strain was tested for its antifungal and antibacterial activity against pathogenic fungi and bacteria. The antifungal activity of S5I4 strain examined by using Aspergillus flavus MD341 (A. flavus) producing aflatoxin B1 as an indicator. The antibacterial activity of S5I4 strain was examined by using Staphylococcus aureus KF 771028 (S. aureus) causing food poisoning as an indicator. This S5I4 strain used to produce the antimicrobial compounds then the extraction, purification and identification of the antimicrobial agents were carried by Gas liquid chromatographic mass spectrometry (GLC-MS). The extract was subjected to GLC-MS to afford 26 peaks corresponding to 26 compounds in ethyl acetate and 20 peaks corresponding to 20 compounds in methylene chloride extract. Most of these compounds are terpenoids (41.30%) terpene alcohols (15.22%), nitrogenous compounds (19.57%) and fatty acids (21.74%) with one miscellaneous group (2.17%) respectively. These compounds produced by S5I4 strain could be mechanism of biocontrol against some fungal diseases.