Background <p>This study investigated variations in vaginal microbiota across female life stages and their correlation with quality of life.</p> Methods <p>This is a cross-sectional study based on the data and sample of 133 women from the outpatient department and health examination center of the hospital between January 2022 and July 2024. They were age-matched within a two-year range and classified into three groups: reproductive (<i>n</i> = 32), perimenopausal (<i>n</i> = 41), and postmenopausal (<i>n</i> = 60). Vaginal swabs were collected for 16&#xa0;S rRNA gene sequencing on the Illumina platform to characterize microbial community structure. General information questionnaires, the Kupperman index, and the Menopause-Specific Quality of Life (MENQOL) questionnaire were administered to evaluate quality of life. Statistical analyses included α/β diversity indices and correlation analysis.</p> Results <p>Significant differences in community diversity and structure were observed among the three groups (<i>P</i> &lt; 0.05). From the reproductive to the postmenopausal stage, vaginal microbial diversity gradually increased, consistent with reduced Lactobacillus dominance, while the abundance of dominant bacteria decreased and the relative abundance of non-Lactobacillus taxa commonly associated with vaginal dysbiosis increased. Perimenopausal symptoms exhibited a worsening trend, accompanied by a notable decline in quality of life, particularly in sexual health.</p> Conclusion <p>Vaginal microbiota differed across reproductive, perimenopausal, and postmenopausal women. Lower Lactobacillus abundance (and higher non-Lactobacillus taxa) was associated with higher Kupperman and MENQOL scores, and Lactobacillus showed a positive association with estradiol.</p>