Objectives: Female sexual and reproductive health is heavily influenced by the levels and ratios of Lactobacilli species and vaginal cytokines. Menopause marks a profound body change as it shifts to a natural and permanent non-reproductive state. Vulvovaginal diseases encompass a broad variety of sexual health conditions. Furthermore, both menopause and vulvovaginal diseases affect vaginal Lactobacilli and cytokine levels. Thus, this study aimed to investigate the correlation between menopause, vulvovaginal diseases, and vaginal Lactobacilli and cytokine levels. Methods: Vaginal swab samples were collected as part of a prospective data bank creation to study vaginal conditions as approved by the Institutional Review Board of Texas Tech University Health Sciences Center, Lubbock, USA. This study utilized 38 samples in this database, which were assigned to the pre-menopausal with no vulvovaginal conditions (n = 20) and post-menopausal with vulvovaginal conditions (n = 18) groups. A real-time polymerase chain reaction was conducted to determine the relative concentration of Lactobacilli species, while cytokine analysis was performed using multiplex enzyme-linked immunosorbent assay immunoassay. The standardized mean difference, multivariate analysis of variance, and permutational unequal variance t test were used for the statistical analysis. Results: Cytokines, interleukin (IL)-6, macrophage inflammatory protein-1α, IL-8, and Lactobacillus iners expression were significantly elevated in the control group compared to the study group (P = 0.03 for the cytokines, P = 0.0194 for Lactobacilli). Conclusions The levels of vaginal cytokine and Lactobacillus profile were significantly different between the pre-menopausal and post-menopausal groups.