OBJECTIVE: Culturomics and 16 S rDNA sequencing were applied to identify lung tumor-resident microorganisms. In vitro characterization revealed potential functions of these cancer-associated microorganisms. METHODS: Eighteen clinical lung cancer (LC) tissues samples were collected. All the samples were cultured by culturomics, and analyzed with 16 S rDNA sequencing. Four isolated isolates belonging to the genus were detected to find out the possible functions in vitro. A549 cells were infected with supernatant of these bacteria, and cell index which reflected cell proliferation was determined by Real-Time Cell Analysis (RTCA). ELISA was employed to detect levels of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in THP-1 cells stimulated with bacterial culture supernatants. RESULT: A total of 12 bacteria were cultured and identified, most of which belonged to the genus. was detected through both methods. In vitro experiment, the cultured strains promoted cellular proliferation, and enhanced the levels of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) as well. CONCLUSION: Bacteria from LC tissues was isolated and identified. Lung tumor-resident microbiota was described. LC tumor-resident bacteria cause tumor development mediated by enhancing tumor cell proliferation and proinflammatory cytokines releasing by macrophages in the tumor microenvironment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12866-025-04237-4.