The aim of this study was to assess the influence of bacterial biofilms in chronic rhinosinusitis (CRS) patients on the clinical outcomes following endoscopic sinus surgery (ESS). This was a prospective, double-blind study. Patients undergoing ESS, because of CRS, were recruited. Overall 80 patients were qualified. For each of these demographic, clinical and radiologic characteristics were recorded. During surgery each patient had at least 7 mucosal specimens taken to assess, using scanning electron microscopy, the possible presence of bacterial biofilms. Prior to mucosa specimen excision, swabs for bacteriological and fungal analyses were taken. Each patient underwent perioperative and follow-up assessment at 3 and 6 months post-ESS. Biofilms were found in 33 (41.3%) patients (study group). From among the 47 patients without the presence of biofilms, 33 (control group) were taken to match the study group in age, gender and clinical characteristics. The intensity of subjective and objective CRS symptoms, as well as patient quality-of-life, did not correlate with the Lund and Mackay score (p > 0.05). Analysis of variance showed that, in the control group or the group as a whole, the intensity of subjective and objective symptoms decreased (p < 0.05), and the quality-of-life increased with time (p < 0.05). In conclusion, biofilm-positive patients tend to have a greater severity of disease preoperatively and continue to have persistent and more severe symptoms post-ESS. This study supports the role of biofilms in maintaining the chronic and recalcitrant nature of CRS. The lack of planktonic bacteria in post-operative sinus swabs does not rule out the presence of bacterial biofilms.