Purpose Lung cancer is the leading cause of cancer death worldwide which includes two main types of carcinoma distinguished in non-small cell lung cancer (NSCLC) involving epithelial cells, and small cell lung cancer (SCLC) affecting neuronal cells and hormone secreting cells. Studies have shown a causal link between inflammation/innate immunity and onset of NSCLC. Hence, the present study aimed to evaluate the diagnostic and prognostic value of Toll-like receptors (TLRs), in particular TLR4 and TLR8 in patients with NSCLC.Methods Patients surgically treated for NSCLC with anatomical resections and patients with benign disease were enrolled. The expression levels of TLR4 and TLR8 were determined by real time PCR and by immunohistochemical analysis in PBMC and in lung tissues, respectively. A preliminary in silico analysis included 1194 arrays from healthy and cancer tissues extracted by Genevestigator database. The association between TLRs gene expression and survival outcome was also investigated.Results Bioinformatics analysis showed a downregulation of TLR4 and TLR8 which positively impacts the survival in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). However, no significant differences between case and control groups were observed for gene expression of TLR4 and TLR8 in PBMC. A positive correlation was found in their expression levels. Interestingly, immunohistochemical analysis showed that the levels of TLR4 and TLR8 were higher in the lung tissues of patients with NSCLC than in the control group either as staining intensity and as positive cells.Conclusions Albeit the precise role of TLRs is not fully defined, this study identified the expression of TLR4 and TLR8 in the pathogenesis of NSCLC. Our data support a possible role for TLR4 and TLR8 in increase overall survival and may serve as a potential biomarker with a diagnostic and prognostic value in NSCLC.