Biologics represent a new therapeutic strategy for severe and recurrent chronic rhinosinusitis with nasal polyps (CRSwNP). Usually, their actual therapeutic effectiveness is assessed by reduction in nasal polyps and/or improvement in nasal symptoms and quality of life. However, these measures do not consider nasal immunophlogosis, which can be evaluated through nasal cytology. The purpose of this study was to assess not only the clinical impact but also the cellular changes in the nasal inflammatory infiltrate observed through nasal cytology of CRSwNP patients treated with Dupilumab for 24 months. Fifty-five CRSwNP patients treated with Dupilumab were collected. Patients were evaluated before starting treatment and at one, three, six, nine months, one year, one and a half years, and two years after the first drug administration. During follow-up visits patients underwent endoscopic evaluation, nasal symptoms and quality of life assessment, complete blood count and nasal cytology. During follow-up, significant improvement was found in Nasal Polyps Score (NPS), nasal patency, olfaction, Sino-Nasal Outcome Test (SNOT-22) score, and Visual Analogue Scale (VAS). Regarding nasal cytology, a reduction in eosinophils and mast cells in the cellular infiltrate was observed over the two-year follow-up period compared to baseline. Dupilumab has demonstrated broad efficacy in the management of CRSwNP from both clinical and cytological findings. Further studies are needed to confirm our findings and evaluate the biologics' impact on nasal mucosal inflammatory cells by nasal cytology with the aim of better identifying each patient's endotype and predicting the response to biologics.