Stroke triggers an intense inflammatory response that could be a consequence of Toll-like receptors (TLRs) activation. However, the clinical significance and the therapeutic possibilities of TLR in stroke is not completely clear. In this study, we analyze the association between the expression of TLR2 and TLR4, inflammatory molecules and endogenous ligands, and clinical outcome of ischemic stroke patients, and we test the potential of TLR2/TLR4 and their endogenous ligands as therapeutic targets. For this purpose, we included 110 patients with ischemic stroke finding that TLR2 and TLR4 are independently associated to poor outcome and correlated with higher serum levels of interleukin (IL)1β, IL6, tumor necrosis factor α, and VCAM1, and that TLR4 was independently associated to lesion volume. In addition, we have developed an in vitro model to test the potential therapeutic value of blocking TLR2/TLR4 or their endogenous ligands. Cultured cells (monocytes and human umbilical vein endothelial cells) were treated with serum from ischemic stroke patients, showing a strong inflammatory response that was blocked when TLR2/4 or cellular fibronectin (cFN) or HSP60 were blocked. In conclusion, TLR2 and TLR4 are associated to outcome in stroke patients and TLR2/4 or their endogenous ligands, cFN/HSP60 could be new therapeutic targets for ischemic stroke.