Lactic acid bacteria are members of probiotic bacteria and their metabolites as biosurfactants (BS_S) emerged as great antibiotics alternatives. The current study aimed to produce, purify and evaluate the antibiofilm effect of BSs produced by Lactobacillus acidophilus isolate against multidrug resistant (MDR) Staphylococcus aureus. BSs were screened by preliminary tests like emulsification assays, oil spreading test and foaming activity. Purification of BS_S was done by silica column chromatography, analyzed by FTIR and Gas chromatography–mass spectrometry. Broth macro-dilution method was employed to find Minimum inhibitory concentration (MIC) of BS_S. Quantitative Reverse Transcriptase Real-Time PCR was to study expression for fib gene implicated in biofilm formation of 30 isolates of MDR S. aureus before and after treating these isolates with three concentrations of sub-MIC of BS_S. Emulsification assay for BS_S was 67%, clear zone (diameter 7 cm) was formed on the surface of petri dish contain oil-water interface. The foaming activity displayed percentage of 60%. FTIR analysis proposed that it composed of glycolipoproteins. GC-MASS showed presence of different biologically compounds as Benzoic acid, n-Hexadecanoic acid, Linoelaidic acid. The MIC value of BS_S against MDR S.aureus isolates  was 0.625 mg/mL. The results revealed that 26 (86.6%) of MDR isolates were positive for biofilm production. Only 4 (13.3%) of isolates produced biofilm after treating with sub-MIC of BS_S. Chemical compounds in BS_S was exposed to the molecular docking analysis against one of biofilm protein (fib protein) of S.aureus. Benzoic_acid possess good affinity toward fib protein (binding energy is 7.5).