Pseudomonas fluorescens is a vital food spoilage bacterium that commonly spoils foods in the biofilm state. Uncovering the targets responsible for biofilm formation and disrupting their function is a promising way to control bacterial biofilms and food spoilage. In this work, using the combination of qRT-PCR and construction of the gene deletion strain, Δtdsr, TonB-dependent siderophore receptor D7M10_RS23410 was, for the first time, proven to play an essential part in the biofilm development of P. fluorescens. By utilizing structure-based virtual screening technology, a natural compound, adenosine monophosphate (AMP), with the highest binding activity to D7M10_RS23410, was obtained as an effective biofilm inhibitor. AMP significantly decreased the cell autoaggregation and biofilm biomass at sub-MIC concentrations (2.5, 1.25, and 0.625 mg/mL), mainly through inhibiting the generation of extracellular polymeric substances (EPS) in the biofilm matrix and promoting the cell motility. Furthermore, AMP was found to form hydrogen bonds with specific amino acid residues and stretched the protein structure of D7M10_RS23410, and this structural alteration undoubtedly interfered with the functionality of the D7M10_RS23410 protein.