Background <p>Increasing evidence has confirmed the existence of resident microbial communities within solid tumors and indicates that the tumor microbiota may represent a novel component of the tumor microenvironment, actively regulating cancer initiation, progression, metastasis, and therapeutic responsiveness. However, the role of tumor microbiota in small cell lung cancer (SCLC) has not been well explored.</p> Methods <p>Tumor samples were collected from 71 patients with SCLC at Shanghai Pulmonary Hospital between 2019 and 2023. A comprehensive analysis of the tumor-resident microbiota in SCLC was conducted using 16&#xa0;S rRNA sequencing. The bacterial communities were profiled, and their correlation with clinical outcomes was assessed.</p> Results <p>A total of 28 phyla, 79 classes, 135 orders, 251 families, and 428 genera were identified, revealing a diverse tumor microbiota in SCLC. It was shown that tumor microbiota varied markedly among patients. SCLC patients with a smoking history exhibited distinct tumor microbiota, with significantly higher abundances of <i>Brevundimonas</i> and <i>Gemmatimonadetes</i>. When patients were stratified by progression-free survival (PFS) into long-PFS (L-PFS) and short-PFS (S-PFS) cohorts, their tumor microbiota segregated distinctly. LEfSe analysis showed that <i>Lactobacillus</i>, <i>Clostridium</i>, <i>Rothia</i>, and <i>Staphylococcus</i> were selectively enriched in the L-PFS group, whereas <i>Stenotrophomonas</i>, <i>Cetobacterium</i>, and <i>Aerococcus</i> dominated the S-PFS group. Kaplan–Meier analysis confirmed that carriage of <i>Lactobacillus</i>, <i>Clostridium</i>, or <i>Staphylococcus</i> was associated with prolonged survival relative to negative status, while positivity for <i>Stenotrophomonas</i>, <i>Cetobacterium</i>, or <i>Aerococcus</i> conferred a reduction in survival. Subsequent response-stratified analysis revealed that <i>Clostridium</i>- and <i>Lactobacillus</i>-positive SCLC was associated with a significantly higher response rate. Conversely, positivity for <i>Methylobacterium</i>, <i>Pelomonas</i>, <i>Ralstonia</i>, <i>Bradyrhizobium</i>, <i>Variovorax</i>, <i>Microbacterium</i>, <i>Comamonas</i>, or <i>Sphingomonas</i> markedly reduced response. The intersection of survival and response results identify <i>Clostridium</i> and <i>Lactobacillus</i> as promising prognostic tumor microbiota markers in SCLC. In vitro assays demonstrated that the <i>Clostridium</i> and <i>Lactobacillus</i> metabolites, butyrate and lactic acid, lacked direct cytotoxicity against SCLC cells. However, in the syngeneic mouse model, systemic supplementation of butyrate or lactic acid significantly potentiated the anti-tumor efficacy of standard chemotherapy. Notably, flow cytometric analysis revealed that this in vivo synergistic effect was closely associated with a profound increase in CD8 + T cell infiltration within the tumor microenvironment. Furthermore, integrating these two tumor microbiota with key clinical variables (sex, age, smoking, stage, radiotherapy), we constructed three models—therapeutic-response, 1-year PFS, and 1-year overall survival—that maintained robust performance in both training and validation cohorts.</p> Conclusions <p>In conclusion, the tumor-resident microbiota constitutes a critical component of the SCLC tumor microenvironment, exerting profound influence on the therapeutic response and patient prognosis. In detail, <i>Clostridium</i> and <i>Lactobacillus</i>—two pivotal tumor-resident taxa significantly linked to enhanced therapeutic responses and favorable prognosis—indicate their potential as predictive biomarkers for treatment outcomes and patient prognosis, and highlight them as candidate targets for microbiome-directed therapeutic strategies against SCLC, which warrants further functional validation.</p> Graphical Abstract <p></p>