Biofilm associated infections are the major contributor of mortality, morbidity and financial burden in patients with a bacterial infection. About 65% of all bacterial infections are associated with the information of bacterial biofilms. Bacterial biofilms not only reduce the efficacy of antibacterial treatment but also increases the threat of developing antibacterial resistance. Recently, our group has discovered the antibacterial activity of Fmoc-phenylalanine (Fmoc-F) and other Fmoc-amino acids (Fmoc-AA). Fmoc-F and other Fmoc-AA showed antibacterial activity due to their surfactant properties. Surfactants are known to eradicate biofilm and enhance antimicrobial activity in biofilm. Thus, in the present study, we evaluated the anti-biofilm activity of Fmoc-F against clinically relevant bacteria. We found that Fmoc-F not only inhibits the biofilm formation in Staphylococcus aureus and Pseudomonas 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 extracellular matrix (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 vancomycin and ampicillin synergistically inhibit biofilm formation. Overall, the study demonstrates the potential application of Fmoc-F and other Fmoc-AA molecules individually as well as in combination as anti-biofilm coating material for treating biofilm associated infections.