ABSTRACTRecently, microbiota dysbiosis in lung cancer has attracted immense attention. Studies on lung microbes are mostly based on sequencing, which has left the potentially functional bacteria with extremely low abundance uncovered. In this study, we characterized and compared the lung and oral cavity microbiotas using culturomics and 16S rRNA gene sequencing. Of the 198 bacteria identified at the species level from bronchoalveolar lavage fluid (BALF) samples,Firmicuteswas predominant (39.90%). Twenty bacterial species isolated from BALF samples were present in at least half of the patients and were also highly abundant in oral samples. Of all isolated strains,StreptococcusandVeillonellawere highly dominant. The abundance ofPrevotellaandVeillonelladecreased from the oral cavity to the lung, whereas that ofPseudomonasincreased. Linear discriminant analysis effect size demonstrated thatPrevotellawas more abundant in the healthy samples than in the cancerous ones, which is in accordance with the isolation ofPrevotella oralisonly from the healthy group using culturomics. Moreover,Gemella sanguinisandStreptococcus intermediuswere isolated only from the non-small-cell lung cancer (NSCLC) group, and 16S rRNA gene sequencing showed that they were higher in the NSCLC than in the small-cell lung cancer group. Furthermore, whileBacillusandCastellaniellawere enriched in lung adenocarcinoma,Brucellawas enriched in lung squamous cell carcinoma. Overall, alterations were observed in the microbial community of patients with lung cancer, whose diversity might be site and pathology dependent. Using culturomics and 16S rRNA gene amplicon sequencing, this study has provided insights into pulmonary and oral microbiota alterations in patients with lung cancer.IMPORTANCEThe relationship between lung microbiota and cancer has been explored based on DNA sequencing; however, culture-dependent approaches are indispensable for further studies on the lung microbiota. In this study, we applied a comprehensive approach combining culturomics and 16S rRNA gene amplicon sequencing to detect members of the microbiotas in saliva and BALF samples from patients with unilateral lobar masses. We found alterations in the microbial community of patients with lung cancer, whose diversity might be site and pathology dependent. These features may be potential bacterial biomarkers and new targets for lung cancer diagnosis and treatment. In addition, a lung and oral microbial biobank from lung cancer patients was established, which represents a useful resource for studies of host-microbe interactions.