The cell fatty acid (FA) composition of adhered and floating cells of two Listeria monocytogenes strains, selected on the basis of the biofilm forming ability, was taken into consideration. Anteiso-15:0, iso-C15:0 and anteiso-17:0 resulted the principal FAs in floating cells. On the other hand the adhered cells of the two strains presented a FAs profile characterized by a prevalence of straight FAs such as hexadecanoic acid (16:0) and octadecanoic acid (18:0). When adhered and free floating cells of the two strains were separately exposed to acid stress, an increase of the individual and total branched FAs was observed particularly in the floating cells of the two strains. The acid stress gave rise in the adhered cells of strain endowed with the lower biofilm forming ability to a relevant intracellular accumulation of straight medium chain FAs. Straight long chain and medium chain free fatty acids (FFAs) were released in the culture supernatants particularly by the strain CH4 endowed with a high biofilm forming ability. The exposure of the latter strain to its own supernatants (conditioned media-CMs) enhanced the C16:0 and C18:0 accumulation in the medium. An unusual quantity of straight medium chain hydrocarbons like n-decane and n-tridecane has been detected particularly in the supernatants of strain. Moreover, long chain hydrocarbons up to nonadecane were released by both strains when exposed to acid stress. Since adhesion is connected with the hydrophobic link of the cells with glass and ionic interactions, these hydrophobic molecules could exercise a key role in the adhesion and de-adhesion mechanisms.