EamB is an inner membrane transporter protein implicated in cysteine and O-acetylserine efflux, as well as in regulating protein synthesis. In this study, we investigated the role of the EamB transporter in biofilm formation of the fish pathogen Aeromonas hydrophila. Deletion of the eamB gene significantly impaired bacterial biofilm formation. Quantitative proteomic analysis identified 616 differentially expressed proteins between the ΔeamB mutant and wild-type (WT) strains, with 308 proteins downregulated and 308 proteins upregulated. Bioinformatic analysis revealed that EamB is involved in critical biological processes, including flagellar assembly, amino acid metabolism, and fatty acid degradation. Microbiological assays demonstrated that exogenous lysine enhanced biofilm formation in the ΔeamB strain but suppressed it in the WT strain, while β-alanine inhibited biofilm formation in the WT strain but had no significant effect on the ΔeamB mutant.