A common feature among Gram-negative bacteria is the production of outer membrane vesicles (OMVs). Once regarded as cell debris or microscopy artifacts OMVs are being increasingly associated with disease status and as bacterial virulence factors. Little is known about OMVs from periodontal pathogens Porphromonas gingivalis, Treponema denticola and Tanerella forsythia, which are strongly associated with the pathology of chronic periodontitis. We have produced highly purified OMVs, using tangential-flow filtration, ultra-centrifugation and Optiprep gradient separation for each of these pathogens and characterised their biophysical/chemical components and evaluated their ability to stimulate the innate immune system via pattern recognition receptors. OMVs from all three bacteria were found to contain a variety of lipids, LPS, peptidoglycan and DNA; in addition P.gingivalis OMVs possess strong protease and haemagglutinating activity. Cryo-TEM and DLS showed that P.gingivalis, T.denticola and T.forsythia OMVs had a size range of; 50-150nm, 30-120nm, 80-250nm, respectively. Using HEK-Blue cell lines stably transfected with TLR and NOD receptors we show that OMVs from all three bacteria strongly stimulated TLR2. Additionally, P.gingivalis and T.forsythia OMVs strongly stimulated TLR4, while T.denticola OMVs induced only a very weak TLR4 response. All OMVs stimulated mild to moderate TLR7, TLR8, TLR9 and NOD2 responses. Overall our results show that P.gingivalis, T.denticola and T.forsythia OMVs contain a variety of PAMPs that may trigger significant inflammatory responses via multiple signalling pathways to contribute to disease progression.