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PURPOSE: Cervical cancer remains a significant public health concern globally and particularly in sub-Saharan Africa, where high rates of HIV infection exacerbate cervical cancer incidence. Understanding the cervical microbiome and its role in cancer progression is essential, especially in regions where both cervical cancer incidence and HIV prevalence are high. This study aimed to characterize the cervical microbiome in women living with HIV (WLWH) and HIV-negative women with squamous cell carcinoma of the cervix in Botswana, compare the microbiome between before and after chemoradiation therapy (CRT) in WLWH, and assess the prognostic value of specific microbial taxa for overall survival (OS) in WLWH. PATIENTS AND METHODS: Cervical samples were collected from women with cervical cancer presenting to one hospital in 2018-2019. Patients' clinical data, including HIV status, were recorded. Microbial composition was analyzed using 16S rRNA gene sequencing. Microbiome diversity and composition were evaluated using alpha and beta diversity metrics. Differential microbial abundance was analyzed using linear discriminant analysis effect size. The association between microbial taxa and OS was explored using Cox proportional hazards regression. RESULTS: = .02), suggesting a more imbalanced microbiome in WLWH. WLWH had higher levels of Parvimonas and members of the Corynebacteriaceae and Micrococcaceae families, suggesting a shift toward a more pathogenic microbiome. In WLWH, CRT did not significantly alter overall microbial diversity. However, Lactobacillus and Sutterella were enriched before treatment, reflecting a less pathogenic microbiome, whereas Ruminococcus and Phascolarctobacterium and the families Caulobacterales and Flavobacteriia were enriched after treatment, reflecting microbial adaptations to the altered immune and treatment environment. Notably, higher levels of Flavobacteriia after CRT were independently associated with worse OS in WLWH. CONCLUSION: Microbiome profiles differ between WLWH and HIV-negative women with cervical cancer in Botswana. The microbiome might have prognostic significance. Future research is needed to better understand the significance of the microbiota in cervical cancer progression and treatment outcomes and the potential role of microbiome-targeted interventions.