Objective: The Eustachian tube (ET) is essential for middle ear ventilation. Chronic obstructive ET dysfunction may lead to chronic otitis media and cholesteatoma, but its pathophysiology remains unclear. Commensal bacterial colonization of the ET has not previously been studied in healthy individuals or affected patients. Study Design and Patients: Prospective cohort study was conducted at a tertiary academic referral center including 13 patients with chronic obstructive ET dysfunction and 37 controls. Between 2017 and 2020, intraoperative tissue samples and swabs were collected using a contamination-minimized Yankauer suction technique. Samples underwent conventional culture diagnostics, microbiome analysis via 16S rDNA sequencing, and histopathological examination. Results: Culture identified 552 bacterial strains, with greater diversity in controls (34 genera) than in patients (19 genera). Microbiome analysis confirmed a larger core microbiome in controls. Rothia was more abundant in patients, whereas Veillonella predominated in controls. Genera such as Rothia and Gemella may be linked to microbial alterations in chronic ET dysfunction. Conclusion: This first combined culture-based and molecular analysis using a novel sampling-technique demonstrated reduced bacterial diversity in chronic ET dysfunction patients, resembling patterns seen in other chronic respiratory morbidities. Future studies should incorporate complementary sampling approaches and cutting-edge sequencing technologies, additionally assessing viral etiologies.  Kulcsszavak: Eustachian tube, microbiome, chronic obstructive ET dysfunction