BackgroundAirway microbiota in asthma-COPD overlap (ACO) remains unknown. ObjectiveThis study with ACO-enriched population aimed to clarify airway microbiota in ACO and in mixed granulocytic inflammation, often detected in ACO and chronic airway diseases. MethodsThis is an observational cross-sectional study. Patients with asthma with airflow limitation, ACO and COPD were enrolled. Blood tests, pulmonary function, exhaled nitric oxide and sputum tests were conducted. Sputum microbiota was evaluated using the 16S rRNA gene sequencing technique. ResultsA total of 112 patients (13 asthma, 67 ACO, 32 COPD) were examined. There were no significant differences in α-diversity among the three diseases. The relative abundances of phylum Bacteroidetes, class Bacteroidia, and genus Porphyromonas were associated with decreased eosinophilic inflammation, and were significantly lower in ACO than in COPD. In a comparison of sputum inflammatory subtypes, the proportion of Haemophilus was numerically highest in the mixed granulocytic subtype, followed by the neutrophilic subtype. Likewise, the proportion of Haemophilus was the highest in the intermediate-high (2%–8%) sputum eosinophil group and lowest in the severe (8%≤) eosinophil group. Clinically, Haemophilus proportion was associated with sputum symptoms. Finally, the proportion of Streptococcus was associated with higher blood eosinophil counts and severest airflow limitation. ConclusionBacteroidia and Porphyromonas abundances in sputum are associated with the eosinophil-low phenotype, and ACO may be characterised by a decrease in these taxa. A mild elevation of sputum eosinophil does not preclude the presence of Haemophilus, which should be noted in the management of obstructive airway diseases.