In recent years, the incidence of female infertility has risen sharply, which is affected by many factors. It was recognized that female reproductive tract microbes play a role in the process of female conception. If the reproductive tract microbes could solve a certain proportion of infertility, it would certainly reduce the pain and economic burden of many patients. The objective of this study was to investigate the microbial community composition of follicular fluid in infertile patients and its potential impact on infertility.Follicular fluid from 49 primary infertility and 52 secondary infertility patients was collected by a negative pressure needle, and the microbiota was analyzed by 16S rDNA sequencing.It was found that Lactobacillus, especially L. crispatus, might have a positive effect on female pregnancy. Considering the presence or absence of male factors and different body mass indices, L. iners might inhibit female pregnancy. However, L. iners seemed to play a positive role in egg maturation, while Gardnerella and Cutibacterium acnes might have a negative effect on female pregnancy.This study suggested the potential role of Lactobacillus in follicular fluid in improving female infertility and provided a theoretical basis for the future microbiological treatment of female infertility.© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.