Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll-Interleukin-1 receptor resistance (TIR) domains. For all TLRs, except TLR3, recruitment of the adapter, MyD88, to the TIR domains results in downstream signaling that culminates in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate diseases caused by TLR-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a "pocket" adjacent to the highly conserved P681 and G682 residues of the BB loop. Using "Computer-Aided Drug Design" (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this "BB loop pocket" and, potentially, disrupt TLR2 signaling. In silico screening identified 149 lead compounds and 20 FDA-approved drugs based on their predicted ability to bind in the BB loop pocket. These were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. In this screen, C16H15NO4 ("C29") was identified as a potential TLR2 inhibitor. C29, and a derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages induced by synthetic and bacterial TLR2 agonists. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6 signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle for this novel CADD approach for the identification of inhibitors of TLR signaling.