Introduction: Bacteria express various pathogen-associated molecular patterns which are recognized by pattern recognition receptors (PRR). PRRs are expressed throughout all immune cells and divided into different groups such as Toll-like receptors (TLR). TLR8 is a sensor of single-stranded RNA, sensing RNA degradation products from viruses or bacteria. It is highly expressed by monocytes, yet its contribution to recognition of bacteria is poorly understood. Goals:The aim of this study was to examine the role of TLR8 for the recognition of whole, viable bacteria in human cell line, thus analysing the importance of bacterial RNA/TLR8 recognition when, during infection, a plethora of different PAMPs is present. The study explored the question of non-redundancy of bacterial RNA mediated TLR8 stimulation. Method:Experiments were conducted with WT and TLR8-defecient BLaER1 cells, which can be transdifferentiated into monocytes. WT and knock-out (KO) cells were infected with different living bacteria. After 1 hour of infection, bacteria were killed by gentamycin and cells were cultivated for another 15 hours. Secreted cytokines in supernatants were quantified. In parallel, we conducted live infections in primary human PBMC pre-treated with the TLR8 inhibitor CU-CPT9a. Results:We identified several bacteria that showed reduced cytokine secretion when TLR8 was missing (BLaER1 KO) or inhibited (PBMCs). Most of them were Gram-positive bacteria including Bacillus cereus. Further, we found Mycoplasma pneumoniae to be dependent on TLR8, indicating that bacterial RNA is a significant PAMP. Reduction in cytokine production in KO cells showed a wide range with some bacteria showing almost no cytokine induction at low MOIs. Gram-negative bacteria, usually recognized via TLR4, surprisingly showed dependence on TLR8 in cases like Haemophilus influenzae. Conclusion: Altogether this highlights the importance of TLR8 for recognition of whole bacteria regardless of the Gram strains and its importance in innate immune response. The study identifies good candidates for further research on bacterial RNA recognition during infection to unravel the complexities of host-pathogen interaction.