This study’s aim was to determine the identity of antibacte-rial compounds produced by Pseudomonas aeruginosa strain UICC B-40 and describe the antibacterial compounds’ me-chanisms of action for damaging pathogenic bacteria cells. Isolation and identification of the compounds were carried out using thin layer chromatography (TLC), nuclear mag-netic resonance (NMR) spectroscopy and liquid chromato-graphy mass spectrometry (LC-MS) analyses. Antibacterial activity was assayed via minimum inhibitory concentration (MIC) and the antibacterial compound mechanism was ob-served morphologically through scanning electron micros-copy (SEM). This study successfully identified the (2E,5E)- phenyltetradeca-2,5-dienoate antibacterial compound (mole-cular weight 300 g/mol), composed of a phenolic ester, fatty acid and long chain of aliphatic group structures. MIC values for this compound were determined at 62.5 μg/ml against Staphylococcus aureus strain ATCC 25923. The mechanism of the compound involved breaking down the bacterial cell walls through the lysis process. The (2E,5E)-phenyltetradeca- 2,5-dienoate compound exhibited inhibitory activity on the growth of Gram-positive bacteria.