Purpose <p>We aimed to evaluate vaginal microbiota distribution before and after laser therapy for gynecologic patients with stress urinary incontinence (SUI) and genitourinary syndrome of menopause (GSM).</p> Methods <p>Fifty patients undergoing CO₂ vaginal laser treatment were enrolled. The primary outcomes included UDI-6, IIQ-7, OABSS, and VSQ-21 scores before and at 1, 2, and 3 months after laser treatment, as well as vaginal microbiota diversity before and 1 month after laser treatment.</p> Results <p>UDI-6, IIQ-7, OABSS, and VSQ-21 scores for patients who received CO₂ laser therapy all showed significant improvement at 1, 2, and 3 months post-treatment. Vaginal pH showed no significant change after laser treatment. Overall, 91 samples were successfully sequenced. Alpha diversity showed significant differences in the menopause (<i>p</i> = 0.034) and GSM (<i>p</i> = 0.004) groups, but not between pre- and post-laser treatment samples. PERMANOVA revealed differences in microbiota composition associated with menopause (<i>p</i> = 0.009) and SUI (<i>p</i> = 0.026). Beta diversity analysis showed no significant treatment effect due to high inter-individual variability. The six most abundant vaginal bacterial genera included <i>Lactobacillus</i> (40.4%), <i>Gardnerella</i> (8.4%), <i>Prevotella</i> (7.4%), <i>Escherichia</i>-<i>Shigella</i> (6.0%), <i>Streptococcus</i> (5.6%), and <i>Bifidobacterium</i> (3.2%), with changes in <i>Lactobacillus</i> dominance observed in some patients post-treatment. Network analysis revealed <i>that Gardnerella</i> disappeared post-laser treatment, as <i>Lactobacillus</i> increased in abundance.</p> Conclusion <p>CO₂ laser treatment may improve SUI and GSM, and be associated with shifts in microbiota.</p>