Bacteria thrive in biofilms embedding in the three-dimensional extracellular polymeric substances (EPS). Functional Amyloid in Pseudomonas (Fap), a protein in EPS, efficiently sequesters polycyclic aromatic hydrocarbons (PAHs). Present study reports the characterization of Fap fibrils from Pseudomonas aeruginosa PFL-P1 and describes the interaction with pyrene to assess the impact on pyrene degradation. Overexpression of fap in E. coli BL21(DE3) cells significantly enhances biofilm formation (p < 0.0001) and amyloid production (p = 0.0002), particularly with pyrene. Defibrillated Fap analysis reveals FapC monomers and increased fibrillation with pyrene. Circular Dichroism (CD), Fourier Transform Infrared Spectroscopy (FTIR), and X-ray Diffraction (XRD) unveil characteristic amyloid peaks and structural changes in Fap fibrils upon pyrene exposure. 3D-EEM analysis identifies a protein-like fluorophore in Fap fibrils, exhibiting pyrene-induced fluorescence quenching. Binding constants range from 5.23 to 7.78 M−1, with ΔG of −5.10 kJ mol−1 at 298 K, indicating spontaneous and exothermic interaction driven by hydrophobic forces. Exogenous Fap fibrils substantially increased the biofilm growth and pyrene degradation by P. aeruginosa PFL-P1 from 46 % to 64 % within 7 days (p = 0.0236). GC–MS identifies diverse metabolites, implying phthalic acid pathway in pyrene degradation. This study deepens insights into structural dynamics of Fap fibrils when exposed to pyrene, offering potential application in environmental bioremediation.