The aim of this study was to investigate the partly membrane properties mechanism of action of linoleic acid (LA) against Lactobacillus acidophilus F0221. F0221 strain was treated with LA at different concentrations range from 0.1 to 10 g/L. Inhibition of cells growth was measured by counting the viable cell counts, alteration of permeability was assessed by measuring the leakage of 260 nm absorbing materials and the Methylene Blue Dye. Change of membrane fluidity was measured by 1, 6-diphenyl-1, 3, 5-hexatriene fluorescence. The effects of LA concentration on CLA production were quantified by measuring c9, t11 CLA levels in cultures of F0221 strain through gas chromatography (GC) and the effects of LA pre-treatment on cells growth were observed. The treatment of F0221 cells with LA at concentrations lower 1 g/L was not significant altered cell counts, whereas at LA concentrations of between 1 and 10 g/L decreased the cell counts, significantly. Compared with control, cell membrane permeability and membrane fluidity was also increased gradually when F0221 strain was cultured with 1 to 10 g/L LA. Additionally, conversion rate of LA into CLA was decreased in a dose dependent manner in the presence of 1.0 to 10 g/L LA and pre-treatment of LA inhibition cells growth with the increase of LA concentration. Date from this study support the hypothesis that LA exerts antibacterial actions by altering membrane properties and compromising membrane associated function.