This study investigated differences in reproductive outcomes and vaginal microbiota profiles between two endometrial preparation protocols—letrozole (LE) combined with human menopausal gonadotropin (HMG) and hormone replacement therapy (HRT) with gonadotropin-releasing hormone agonist (GnRH-a) pretreatment—in women with endometriosis undergoing frozen embryo transfer (FET). Following 1∶1 propensity score matching, a total of 770 FET cycles were analyzed. No statistically significant differences were observed in live birth rates or clinical pregnancy rates between the two groups. However, the LE + HMG group showed a nonsignificant trend toward a lower miscarriage rate (13.7% vs. 19.8%, P = 0.070), as well as lower observed rates of cesarean delivery (64.9% vs. 75.4%, P = 0.020) and hypertensive disorders of pregnancy (4.8% vs. 10.1%, P = 0.039). Previous evidence suggests an association between GnRH-a treatment and reproductive tract microbiota composition. Given the clinical and ethical constraints of endometrial sampling during FET, vaginal microbiota was evaluated as a non-invasive indicator of lower reproductive tract microbial composition. In the prospective arm, vaginal samples from 55 women in the LE + HMG group and 50 in the GnRH-a HRT group were analyzed using 16S rRNA sequencing and droplet digital PCR. While no significant differences were observed in Lactobacillus or Gardnerella abundance, the GnRH-a HRT group showed higher relative abundances of several low-abundance taxa, such as Escherichia-Shigella, Limosilactobacillus, and Staphylococcus. In conclusion, although both protocols achieved comparable live birth outcomes, the LE + HMG regimen was associated with lower rates of cesarean delivery and hypertensive disorders of pregnancy, while the two protocols exhibited differences in several low-abundance vaginal bacterial taxa. However, longitudinal studies incorporating baseline and repeated sampling are needed to clarify temporal relationships and causality.