Background: Non-hormonal contraception methods have been widely used, but their effects on colonization by vaginal lactobacilli remain unclear. Objective: To determine the association between non-hormonal contraception methods and vaginal lactobacilli on women's reproductive health. Methods: The cross-sectional study included 164 healthy women between 18-45 years of age. The subjects were divided into different groups on the basis of the different non-hormonal contraception methods used by them. At the postmenstrual visit (day 21 or 22 of the menstrual cycle), vaginal swabs were collected for determination of Nugent score, quantitative culture and real-time polymerase chain reaction (PCR) of vaginal lactobacilli. The prevalence, colony counts and 16S rRNA gene expression of the Lactobacillus strains were compared between the different groups by Chi-square and ANOVA statistical analysis methods. Results: A Nugent score of 0-3 was more common in the condom group (93.1%) than in the group that used an interuterine device(IUD) (75.4%), (p = 0.005). The prevalence of H2O2-producing Lactobacillus was significantly higher in the condom group (82.3%) than in the IUD group (68.2%), (p = 0.016). There was a significant difference in colony count (mean +/- standard error (SE), log(10)colony forming unit (CFU)/ml) of H2O2-producing Lactobacillus between condom users (7.81 +/- 0.14) and IUD users (6.54 +/- 0.14), (p = 0.000). The 16S rRNA gene expression (mean +/- SE, log(10)copies/ml) of Lactobacillus crispatus was significantly higher in the condom group (8.09 +/- 0.16) than in the IUD group (6.03 +/- 0.18), (p = 0.000). Conclusion: Consistent condom use increases the colonization of Lactobacillus crispatus in the vagina and may protect against both bacterial vaginosis (BV) and human immunodeficiency virus (HIV).