The chemical screening tests are important tools for the detection of bioactive principles. In this study, gas chromatography−mass spectrometry (GC−MS) was successfully used to identify the biochemical constituents produced by Lactobacillus plantarum and Lactobacillus acidophilus strains. The method is based on liquid-liquid extraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. The GC-MS chromatogram of the extracts of L. plantarum and L. Acidophilus revealed the presence of twenty-two and seventeen chemical compounds respectively with antimicrobial activities. Bioactive compounds as Pentadecanoic acid, Butanoic acid, Eucalyptol, Isopropyl Myristate possess, and 9-Hexadecenoic acid were detected in the extract of L. plantarum. Also, other compounds as 1- Tetradecanol, Isochiapin B, cis-Vaccenic acid, 1,2-Benzenedicarboxylic acid were detected for L. acidophilus. Furthermore, different bioactive compounds with antimicrobial activities as Oleic acid, 1-Hexadecanol, 2-methyl, 2,2- Dideutero octadecanal, 9-Octadecenoic acid were detected in both extracts of L. plantarum and L. Acidophilus. Thus, these probiotics are strong candidates as antimicrobial agents.