Understanding the interactions between the cervico-vaginal microbiome, immune responses, and sexually transmitted infections (STIs) is crucial for developing targeted diagnostic and therapeutic strategies. Although microbiome analyses are not yet standard practice, integrating them into routine diagnostics could enhance personalized medicine and therapies. We investigated the extent to which partial 16S short-read amplicon microbiome analyses could inform on the presence of six commonly encountered STI-causing pathogens in a patient cohort referred for colposcopy, and whether relevant taxonomic or diagnostic discrepancies occur when using vaginal rather than cervical swabs. The study cohort included cervical and vaginal samples collected from women referred for colposcopy at the University Hospital Bonn between November 2021 and February 2022, due to an abnormal PAP smear or positive hrHPV results. 16S rRNA gene sequencing libraries were prepared targeting the V1-V2 and V4 regions of the 16S RNA gene and sequenced on the Illumina MiSeq. PCR diagnostics for common STI-causing pathogens were conducted using the Allplex STI Essential Assay Kit (Seegene, Seoul, Republic of Korea). Concerning the bacterial microbiome, no significant differences were found between vaginal and cervical samples in terms of prevalence of taxa present or diversity. A total of 95 patients and 171 samples tested positive for at least one among