Aeromonas hydrophila is an opportunistic Gram‐negative pathogen that readily attaches to stainless steel to produce a thin biofilm with a complex 3D structure covering 40–50% of the available surface and producing large microcolonies. As A. hydrophila possesses an N‐acylhomoserine lactone (AHL)‐dependent quorum‐sensing system based on the ahyRI locus, the presence of the AhyI protein and C4‐HSL within the biofilm phase was first established by Western blot and AHL biosensor analysis respectively. The ability of the A. hydrophila AH‐1 N strain to form biofilms in a continuous‐flow chamber was compared with isogenic ahyI and ahyR mutants. The ahyI mutant, which cannot produce C4‐HSL, failed to form a mature biofilm. In addition, the viable count of biofilm, but not planktonic phase ahyI mutants, was significantly lower that the parent or ahyR mutant. This defect in the differentiation of the ahyI mutant biofilm could be partially restored by the addition of exogenous C4‐HSL. A mutation in ahyR increased coverage of the available surface to around 80% with no obvious effect upon biofilm microcolony formation. These data support a role for AHL‐dependent quorum sensing in A. hydrophila biofilm development. Exposure of the A. hydrophila AH‐1N biofilm to N‐(3‐oxodecanoyl)homoserine lactone, which inhibits exoprotease production in planktonic cells, however, had no effect on biofilm formation or architecture within the continuous‐flow chamber.