Column experiments were conducted to investigate the transport of aqueous C60 (aqu-nC60), fullerol, silver nanoparticles (NPs) coated with polyvinylpyrrolidone (Ag-PVP) and stabilized by citrate (Ag-CIT) in biofilm-laden porous media. Gram-negative Pseudomonas aeruginosa (PA) and Gram-positive Bacillus cereus (BC) biofilm-laden glass beads were selected to represent the bacterial interfaces NPs might encounter in the natural aquatic environment. The biomass distribution, extracellular polymeric substances (EPS) components, electrokinetic property, and hydrophobicity of these interfaces were characterized, and the hydrophobicity was found to correlate with the quantity of proteins in EPS. The retention of NPs on glass beads coated with bovine serum albumin (BSA) and alginate were also studied. Except for Ag-PVP, the affinity of NPs for porous medium, indicated by attachment efficiency α, increased in the presence of biofilms, BSA and alginate. For hydrophobic aqu-nC60, the larger the proteins/polysaccharides ratio, the larger the α, suggesting the hydrophobic interaction determines the attachment of aqu-nC60 to the collector surface. Uncharged PVP stabilized Ag-PVP by steric repulsion, and the attachment to glass beads was not enhanced by biofilm. The presence of divalent ion Ca2+ significantly hydrophobized biofilm, BSA, and alginate-coated glass beads and further retarded the mobility of aqu-nC60, fullerol, and Ag-CIT; while Ag-PVP was again sterically stabilized.