Background: Bacterial biofilm formation is associated with chronic rhinosinusitis (CRS). However, how bacterial biofilms are related to innate immune response in patients is poorly understood. The aim of this study was to evaluate the prevalence of biofilms in CRS patients and investigate the biofilm-related Toll-like receptor (TLR) and downstream NF-kappaB expression in sinus tissues of patients with CRS. Methods: A total of 31 CRS patients and 11 control patients with other diseases undergoing endoscopic sinus surgery were recruited consecutively. Their random sinonasal tissue specimens were examined for the formation of biofilms by scanning electron microscopy. The relative levels of TLR2, TLR4, and NF-kappaB expression in those sinus tissues were determined by quantitative real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. Results: Bacterial biofilms were detected in 20 of 31 samples from CRS patients but not in a single sample from control patients. The relative levels of TLR2, TLR4, and NF-kappaB expression in sinus tissues from CRS patients were significantly higher than that of controls, and the relative levels of TLR2 and NF-kappaB, but not TLR4, in biofilm-positive sinus tissues were significantly higher than that in biofilm-negative tissues of those CRS patients. Conclusion: Our data indicated that bacterial biofilms were associated with higher levels of TLR2 and NF-kappaB in the majority of sinus tissues from patients with CRS. (Am J Rhinol Allergy 26, 104-109, 2012; doi: 10.2500/ajra.2012.26.3718)