The vaginal microbiota is a critical component of female reproductive and urogenital health, predominantly maintained by Lactobacillus species. These bacteria play a vital role in sustaining a low vaginal pH by producing lactic acid and antimicrobial substances, providing natural defense against pathogenic colonization. The hormonal alterations linked to menopause, especially the reduction in oestrogen levels, significantly influence the composition and stability of the vaginal microbiome, making women vulnerable to infections and inflammation. This investigation sought to assess and contrast the vaginal microbiome composition in women before and after menopause. Using Gram-stain microscopy, conventional culture methods, and molecular techniques, specifically Polymerase Chain Reaction (PCR) and quantitative real-time PCR (qPCR), to assess Lactobacillus abundance and the prevalence of pathogenic bacteria. A comparative cross-sectional study was conducted by comparing two groups of women: 40 premenopausal and 40 postmenopausal, for a total of 80 participants. Vaginal swabs were collected and subjected to Gram staining, culture on selective media for Candida spp., and PCR/qPCR analysis to detect and quantify key bacterial species, including Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae. SPSS Version 25 was employed for conducting. The statistical analysis yielded data viewed significant at p < 0.05. Microscopic observation revealed that 85% of premenopausal women had Nugent scores indicating a healthy Lactobacillus-dominated microbiota, while only 35% of postmenopausal women showed similar results. PCR and qPCR analyses demonstrated a significant reduction in Lactobacillus spp. in postmenopausal women, with Lactobacillus crispatus copy numbers declining from 4.5 × 10¹¹ to 1.2 × 10¹¹ copies/mL. Conversely, pathogenic bacteria such as Gardnerella vaginalis and Prevotella spp. increased significantly in postmenopausal women. Additionally, Candida spp. colonization was high in the postmenopausal group (15%) compare to the premenopausal group (5%). The findings confirm a significant shift in vaginal microbiota composition due to menopausal hormonal changes. The depletion of Lactobacillus spp. and the concurrent rise in pathogenic bacteria in postmenopausal women underscore the need for targeted interventions, such as probiotic therapies or hormone replacement treatments, to restore microbial balance and reduce infection risks.