Background: Biofilm associated infections are the major contributor to mortality, morbidity and financial burden in patients with a bacterial infection. Molecules with surfactant behavior are known to show significant antibiofilm effect against these infections. Thus, newly discovered antibacterial Fmoc-phenylalanine (Fmoc-F) and other Fmoc-amino acids (Fmoc-AA) with surfactant properties, could have potential antibiofilm properties. Objectives: To evaluate and characterize the antibiofilm activity of Fmoc-F and some Fmoc-AA against various clinically relevant bacteria. Methods: Biofilm inhibition and eradication were evaluated by crystal violet staining procedure along with scanning electron microscopy (SEM). Attenuated Total Reflection - Fourier Transform Infrared Spectroscopy (ATR-FTIR), Biochemical assays, and Congo red staining were employed to investigate the mechanism of antibiofilm action. Results: We showed that Fmoc-F not only inhibits the biofilm formation in S. aureus and P. aeruginosa, but also eradicates the already formed biofilms over the surface. Further, Fmoc-F coated glass surface resists S. aureus and P. aeruginosa biofilm formation and attachment, when biofilm is grown over the surface. The mechanistic investigation suggests that Fmoc-F reduces the ECM components such as proteins carbohydrates and eDNA in the biofilm and affect its stability via direct interactions with ECM components and/ or indirectly through reducing bacterial cell population. Finally, we showed that Fmoc-F treatment in combination with other antibiotics such as vancomycin and ampicillin synergistically inhibit biofilm formation. Conclusions: Overall, the study demonstrates the potential application of Fmoc-F and other Fmoc-AA molecules individually as well as in combination as antibiofilm agents and antibiofilm coating material for treating biofilm-associated infections. ### Competing Interest Statement The authors have declared no competing interest.

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