The composition of the vaginal microbiome plays a crucial role in women's health, and disruptions in microbial balance can lead to various health issues. This study aimed to assess the suitability of self-collected diagnostic tampons as a specimen collection device for analyzing the vaginal microbiome using qualitative real-time polymerase chain reaction (rtPCR). Tampon samples were compared to self-collected lower vaginal swabs (LVSs) and clinician-collected 1 cervical swabs in a cohort of 83 female subjects. The tampons used for self-sampling are 100% organic cotton diagnostic tampons (Daye brand, Anne’s Day Ltd). The results showed that the tampon samples detected significantly higher levels of Total Bacterial Mass (TBM), compared to cervical and vaginal swabs (7.24, 6.87 and 7.25 log (GE/ml) respectively, p = 0.0002), Lactobacillus spp. (7.13, 6.76 and 7.16 log (GE/ml) p = 0.0019), and Ureaplasma spp. (4.12, 3.78 and 4.23 log (GE/ml), p = 0.0279), compared to cervical and vaginal swabs. There was no significant difference in the detection of Candida spp. (3.92, 3.83 and 4.27 log (GE/ml), p = 0.3346) or Gardnerella vaginalis (4.43, 4.29 and 4.46 log (GE/ml), p = 0.2293). Overall, the diagnostic tampons showed promise as a convenient and effective self-collection tool for analyzing the vaginal microbiome. This paper highlights the importance of understanding and maintaining a healthy vaginal microbiome for women's health and provides valuable insights into new methods for sample collection towards detecting genital infections.