The present study focuses on the chemical composition of the ethyl acetate organic fraction derived from cell-free culture of thermophilic Bacillus licheniformis MK027019 by GC-MS. In addition, the antibacterial activity of the organic fraction against two multidrug-resistant microorganisms is tested using the Radial Diffusion Assay technique. GC-MS analysis revealed that thermophilic B. licheniformis MK027019 produced five major groups of secondary metabolites based on their retention time, molecular formula and molecular weight. These groups were: [Germacrene-D: 14.70%], saturated hydrocarbon [Eicosane: 12.13%, Heneicosane: 3.17%], fatty acid methyl ester [hexadecanoic acid methyl ester: 6.51%, eicosanoic acid methyl ester: 0.78%], alkane [n-Tridecane: 4.39%] and quinoline derivative [8-Phenyl-isoquinoline: 3.51%]. This organic fraction is effective against the multidrug-resistant bacterium Pseudomonas aeruginosa. The presence of Germacrene-D and quinoline derivative are significant in this finding, and to our knowledge, this is the first time these compounds have been detected in thermophilic Bacillus licheniformis.