Background: The emerging concept of intracellular pathogens such as Staphylococcus aureus playing a role in chronic rhinosinusitis (CRS) has led to the development of numerous imaging techniques for their identification. Traditional methods of bacterial culture are not effective at localizing bacteria to the surface or within tissue samples. The aim of this study was to develop and validate a novel imaging technique using confocal scanning laser microscopy (CSLM) coupled with a fluorescence in situ hybridization (FISH) probe and nucleic acid counterstain (propidium iodide [PI]) that allows for simultaneous analysis of S. aureus intracellular status and surface biofilm within whole mucosal samples. Methods: A prospective study was performed including 17 patients undergoing endoscopic sinus surgery for CRS. Tissue samples were analyzed with both CSLM-FISH/PI and immunohistochemistry (IHC) for intracellular S. aureus status. Results: Using CSLM-FISH/PI intracellular S. aureus was identified in 9/17 (47%) patients and in 7/17 (39%) using IHC. Surface biofilm can be identified with CSLM-FISH/PI in the same piece of tissue; however, deeper imaging to the submucosa is impossible. IHC showed submucosal bacteria in three patients. Conclusion: Both CSLM-FISH/PI and IHC are complementary techniques that can be used to identify intracellular S. aureus. CSLM-FISH/PI allows for the simultaneous detection of intracellular status and surface biofilm within the tissue analyzed. IHC has a role in the identification of intracellular and submucosal S. aureus within these tissues. Additional investigation is required to identify the true pathogenic nature of intracellular organisms as well as any relationship to surface biofilm status.