Objective(s):Serratia marcescens is one of the nosocomial pathogen. The ability to form biofilm is an important feature in the pathogenesis of S. marcescens. The aim of this study was to determine the anti-adhesive properties of a biosurfactant isolated from Lactobacillus acidophilus ATCC 4356, on S. marcescens strains. Materials and Methods: Lactobacillus acidophilus ATCC 4356, was selected as a probiotic strain to produce biosurfactant. Anti-adhesive activities was determined by pre-coating and co- incubating methods in 96-well culture plates. Results: The FTIR analysis of derived biosurfactant revealed the composition as protein component. Because of the release of such biosurfactants, L. acidophilus was able to interfere with the adhesion and biofilm formation of the S. marcescens strains. In co- incubation method this biosurfactant in 2.5 mg/ml concentration showed anti-adhesive activity against all tested strains of S. marcescens (P