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Biosurfactants derived from different microbes are an alternative to chemical surfactants, which have broad applications in food, oil, biodegradation, cosmetic, agriculture, pesticide and medicine/pharmaceutical industries. This is due to their environmentally friendly, biocompatible, biodegradable, effectiveness to work under various environmental conditions and non-toxic nature. Lactic acid bacteria (LAB)-derived glycolipid biosurfactants can play a major role in preventing bacterial attachment, biofilm eradication and related infections in various clinical settings and industries. Hence, it is important to explore and identify the novel molecule/method for the treatment of biofilms of pathogenic bacteria. In the present study, a probiotic Lactobacillus rhamnosus (L. rhamnosus) strain was isolated from human breast milk. Firstly, its ability to produce biosurfactants, and its physicochemical and functional properties (critical micelle concentration (CMC), reduction in surface tension, emulsification index (% EI24), etc.) were evaluated. Secondly, inhibition of bacterial adhesion and biofilm eradication by cell-bound biosurfactants from L. rhamnosus was performed against various biofilm-forming pathogens (B. subtilis, P. aeruginosa, S. aureus and E. coli). Finally, bacterial cell damage, viability of cells within the biofilm, exopolysaccharide (EPS) production and identification of the structural analogues of the crude biosurfactant via gas chromatography–mass spectrometry (GC–MS) analysis were also evaluated. As a result, L. rhamnosus was found to produce 4.32 ± 0.19 g/L biosurfactant that displayed a CMC of 3.0 g/L and reduced the surface tension from 71.12 …