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The antibacterial activity and mechanism of <i>Pinus densiflora</i> extracts against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> were investigated. The growth inhibition tests of paper diffusion and optical density exhibited that the extracts have potent antibacterial potentials against foodborne pathogens. The measurement of membrane fluidity by fluorescence polarization has indicated that one of the antibacterial mechanisms involves the disruption of membrane integrity resulting in an increase in the membrane fluidity in both of <i>E. coli</i> and <i>S. aureus</i>. The alteration of fatty acid composition was accompanied by the disturbance of membranes thus shifting the proportion of saturated verses unsaturated fatty acids or trans fatty acids from 1.27:1 to 1.35:1 in <i>E. coli</i> and 1.47:1 to 2.31:1 in <i>S. aureus</i>, most likely to compensate for the increased membrane fluidity by means of a higher proportion of saturated fatty acids which is known to render rigidity in membranes. Realtime q-PCR (polymerase chain reaction) analysis of fatty acid synthetic genes and bacterial stress genes revealed that there was minimal influence of <i>P. densiflora</i> extracts on fatty acid genes except for <i>fab I</i> and the stress <i>rpos</i> in <i>E. coli</i>, and relatively greater impact on fatty acid genes and the stress <i>sigB</i> in <i>S. aureus</i>.