Quorum sensing (QS) or cell-to-cell communication is a process by which bacteria produce and detect signal molecules and thereby coordinate their behavior in a cell-density dependent manner. Two main QS systems can be distinguished: the acylhomoserine lactone (AHL) and the autoinducer-2 (AI-2). The sequenced genome of the marine bioluminescent Shewanella woodyi MS32 contains genes coding for these two main QS systems. This work aims at identifying the QS communication systems present in this bacterium and understanding its role in luminescence production and biofilm formation. The objective is also to find molecules that interfere with QS processes to inhibit adhesion and biofilm formation. By coupling bacterial biosensor assays and high resolution liquid chromatography mass spectrometry (LCMS), we showed that S. woodyi synthesized both a N-octanoyl-l-homoserine lactone (C8-HSL) and the AI2 QS molecule. However, only HSL molecules influence the luminescence of S. woodyi in particular oxo and hydroxy derivatives. Several commercial molecules, identified for their inhibitory effect on the AI2 QS system of Vibrio harveyi Inhibited the luminescence of S. woodyi and reduced adhesion and biofilm formation, which led to the conclusion that AI-2 is not likely used as a quorum sensing signal and that LuxS is involved in the activated methyl cycle (AMC) in S. Woodyi. This study is the first documentation that characterizes the QS system in S. Woodyi. Long side chains AHLs biosensor (Agrobacterium tumefaciens A 136) Short side chains AHLs biosensor (Chromobacterium violaceum CV 026) WT S+ R+ A136 S-R+ S. woodyi A136 S-R+ Various AHLs modulate S. woodyi luminescence S. woodyi produces AI2, however luminescence is under the control of the AHL QS system. Bioluminescence induction in the AI2 biosensor Vibrio harveyi MM32 after addition of Marine Broth (MB, negative control), culture supernatant of V. harveyi WT BB120 as a source of exogenous AI2 (positive control) or culture supernatant of S. woodyi. S. woodyi produces AI2 Bioluminescence induction in S. woodyi after addition of Marine Broth (MB, negative control), culture supernatant of V. harveyi BB475 as a source of AI2, culture supernatant of S. woodyi or culture supernatant of V. harveyi WT BB120 as a source of exogenous AI2 and 3OH C4-HSL. Luminescence of S. woodyi is mediated by the AHL QS system and is not under the control of the AI2 QS system. This observation is in good agreement with the absence of the luxPQ genes in the genome of S. woodyi.