The lungs are now recognized to harbor a low biomass and a diverse microbial community which is altered in lung diseases. However, whether lung microbiota (LM) alteration is a cause or a consequence of lung pathologies is still a subject for debate. Bronchomalacia (BM), that is defined as a regional to diffuse dynamic airway collapse of bronchi causing airflow limitation, has been inconsistently associated with bacterial infection and it is unclear whether bacteria contribute to the disease pathogenesis and progression or not. In this study, we assessed the LM in dogs with reported bronchomala- cia at bronchoscopy (BM-dogs; n = 25) in comparison with healthy dogs (H-dogs; n = 37). Dogs with primary or secondary bacterial and parasitic infection were excluded based on Baerman test and bronch- oalveolar lavage fluid (BALF) cytology, culture and Bordetella bronchi- septica, Mycoplasma cynos, Angiostrongylus vasorum and Crenosoma vulpis qPCRs; as were dogs receiving antimicrobial drug in the week before sampling. LM was characterized using DNA extracted from naïve BALF subjected to 16S rRNA amplicon sequencing. Bacterial load was measured in duplicate using qPCR targeting the V2-V3 region of the 16S rRNA gene. Bacterial composition, load and ecologi- cal parameters were calculated and compared between BM-dogs and H-dogs using MOTHUR v3, STAMP v2.1.3, XLSTAT v4.1 and R vegan package v 3.6.2. Among the 25 BM-dogs, based on BALF analysis, 3 had concomitant eosinophilic and 15 neutrophilic infiltrations, while others had a nor- mal analysis. No significant differences between bacterial loads (P = 0.075) were found between groups. In BM-dogs, the LM was dominated by Conchiformibius, Streptococcus, Staphylococcus, Chryseo- bacterium and Serratia genera and some genera of the Pasteurellaceae family. Linear discriminant analysis effect size revealed that the LM in BM-dogs was enriched in 19 genera present in lower proportion in H-dogs and commonly found in upper airways or digestive microbiota. Permutational multivariate analysis of variance detected a significant difference in LM between BM-dogs and H-dogs (P = 0.001). Finally, decreased bacterial alpha-diversity (median and interquartile range of 8.03 (2.81 – 10.72) vs. 9.14 (5.51 – 15.08); P = 0.037) and evenness (0.06 (0.03 – 0.10) versus 0.09 (0.06 – 0.18); P = 0.011) were observed in BM-dogs compared with H-dogs. This comparative study provides evidence for alteration of the LM in BM-dogs. Because of the increase in genera from upper airways and digestive microbiota, altered LM might be secondary to an increase in the micro-aspiration rate which is suggested in that disease. Further studies are warranted to determine if such bacteria have a role in dis- ease onset or progression.