In women receiving assisted reproductive treatment, intrauterine lactobacilli dominance has been associated with higher rates of pregnancy achievement. This randomized controlled trial conducted in the fertility clinic of the university hospital from 7 August 2019 to May 2021, aimed to compare the clinical outcome of embryo transfer in frozen-thaw cycles with Lactobacillus supplementation prior to embryo transfer and the standard treatment. A total of 340 infertile women underwent randomization. The biochemical and clinical pregnancy rates were comparable between the groups (39.9 and 34.2% in the study group vs. 41.8 and 31.7% in the control group); however, the miscarriage rate was significantly decreased in the study group (9.5 vs. 19.1%, respectively, p = 0.02), [OR = 0.44, 95% CI (0.23, 0.86)]. Among 49 women diagnosed with bacterial vaginosis, the live birth rate in the study group was higher than the control group (42.31 vs. 26.09%, p = 0.23), [OR = 2.08, 95% CI (0.62, 6.99)]. In the blastocyst transfer group (n = 206), the live birth rate was significantly higher in the study group than in the control group (35.71 vs. 22.22%, p = 0.03) [OR = 1.9, 95% CI (1.05, 3.59)]. Therefore, intravaginal lactobacilli supplementation before embryo transfer in the frozen-thaw cycle did not improve the biochemical and clinical pregnancy rate in the general population but significantly reduced the miscarriage rate.Trial Registration: TCTR20190429001 (29/04/2019) @ www.thaiclinicaltrials.org .