Abstract Toll-like receptors (TLRs) are pattern recognition receptors (PRR) that recognize molecular structures on pathogens and activate host defenses. Although much is known about specific bacterial components that activate TLRs, few studies have addressed the question of which TLRs are involved in immune activation by live bacteria. Here, we demonstrate that live Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitidis, the three principal causes of bacterial meningitis, use distinct sets of TLRs to trigger the inflammatory response. Using human embryonic kidney 293 cell lines, each overexpressing one type of TLR, we found that S. pneumoniae triggered activation of the transcription factor nuclear factor-κB and expression of interleukin-8, only in cells expressing TLR2 or -9. The same response was evoked by H. influenzae in cells expressing TLR2 or -4 and by N. meningitidis in cells expressing TLR2, -4, or -9. It is interesting that the ability of S. pneumoniae and N. meningitidis to activate TLR9 was severely attenuated when bacteria had been heat-inactivated prior to stimulation of the cells. In human peripheral blood mononuclear cells, we blocked TLR2, -4, or -9 and confirmed the essential role of these TLRs and also identified differential functions of TLRs in activation of the inflammatory response. Collectively, we here demonstrate that S. pneumoniae, H. influenzae, and N. meningitidis each activate several TLRs in species-specific patterns and show that infection with live pathogens may lead to activation of PRR not targeted by inactivated bacteria.