The ability of surfactants obtained from three Lactobacillus acidophilus strains to inhibit Staphylococcus aureus and S. epidermidis biofilms was evaluated. Their influence was determined on bacterial initial adhesion, biofilm formation and dispersal using MTT-reduction assay, confocal laser scanning microscopy and image PHLIP analysis. The number of adhering S. aureus and S. epidermidis cells after a 3-h co-incubation with biosurfactants was reduced by 5-56 % in a strain-and dose-dependent manner. S. epidermidis-and, to a lower extent, in S. aureus-biofilm formation was also inhibited in the presence of the tested surfactants. The addition of surfactants to preformed mature biofilms accelerated their dispersal, and changed the parameters of biofilm morphology. The L. acidophilus-derived surfactants inhibit bacterial deposition rate and biofilm development (and also its maturation) without affecting cell growth probably due to the influence on the cell-surface hydrophobicity of staphylococci.