Background: The Eustachian tube is essential for middle ear health, ensuring pressure equalization, ventilation, and protection from pathogens. Smoking has been linked to altered Eustachian tube function, affecting mucociliary activity and middle ear ventilation. Aims/Objectives: This study evaluates smoking's impact on Eustachian tube function using tympanometry, nasal mucociliary clearance testing, and the Eustachian Tube Dysfunction Questionnaire. Material and Methods: Healthy adults aged 20-50 years with intact tympanic membranes, no upper respiratory infections, or nasal pathologies were included. Participants were divided into smokers (subgrouped by smoking history: 5-10, 10-20, and >20 pack-years) and non-smokers. Tympanometry, mucociliary clearance testing, and the questionnaire were administered. Results: Among 276 participants, significant differences in questionnaire scores, compliance, middle ear pressure, and mucociliary clearance times were observed between groups (p < 0.01, p = 0.02, p < 0.001, and p = 0.009, respectively). Ipsilateral acoustic reflexes differed significantly between groups (p < 0.005 and p = 0.049). Mucociliary clearance times were notably prolonged in the >20 pack-years group compared to the 5-10 pack-years group (p = 0.012). Conclusions and Significance: Smoking adversely affects Eustachian tube function, as evidenced by tympanometric, mucociliary, and questionnaire assessments.