The bioactive chemical constituents of secondary metabolites from probiotic Bacillus amyloliquefaciens SW19 were isolated from midgut silkworm. These secondary metabolites had antibacterial activity against Bacillus thuringiensis SW2 which caused silkworm bacterial flacherie. The bioactive component of the ethyl acetate extract from B. amyloliquefaciens SW19 was analyzed by using GC-MS. GC-MS is a simple, rapid and accurate method for analyzing metabolites. GC-MS finger print had better resolution and estimation of active constituents with reasonable accuracy in a shorter time. From the study, 15 components in secretion were confirmed. They showed a variety of function like antioxidants, antibacterial and antimicrobial agents. Finally, these compounds were purified by TLC and identified for pure compound by GC-MS after applying their activity on Bacillus thuriengiensis SW2. The first one phenol, 2, 4-bis (1, 1-dimethylethyl) and the second one 9-Octadecenoic (Z)-2, 3-dihydroxylpropyl ester were identified by GC/MS with molecular weight 278.511 and 354.539 g/mol, respectively. These compounds had clear antimicrobial activity against B. thuriengiensis SW2.