The female reproductive tract harbours its microbial communities (microbiota) and its composition has been associated with reproductive functions in health and disease. Endometrial microbiome (genomes of microbes) studies are in agreement that uterus possesses higher bacterial diversity and richness than the vagina (where Lactobacillus is dominating) but the knowledge of the Fallopian tubes (FT) microbiomal composition is lacking, especially in fertile women in disease-free conditions. Our study included 24 women with confirmed fertility having benign uterine pathology submitted to abdominal hysterectomy or opting for contraceptive method by tubal ligation at Hospital Clínico Universitario Virgen de la Arrixaca de Murcia (HCUVA). The microbiome of FT and endometrial samples was analyzed by 16S rRNA gene sequencing. We detected distinct microbial community profiles in the endometrial and FT samples indicating that the upper reproductive tract harbors an endogenous microbiome and in big part these two sites share the microbial profile (69% of the detected taxa). Nevertheless, 17 bacterial taxa were exclusively detected in the FT that included Enhydrobacter, Granulicatella, Haemophilus, Rhizobium, Alistipes y Paracoccus, among others, while 10 were found only in the endometrium, including Klebsiella, Olsenella, Oscillibacter and Veillonella (FDR <0.05). Further, our study shows that the endometrial collection method influences the findings, where Lactobacillus dominated in the samples obtained transcervically (vaginal contamination is suspected), while Acinetobacter, Arthrobacter, Coprococcus, Methylobacterium, Prevotella, Roseburia, Staphylococcus, Streptococcus were more abundant when sampling was performed directly from the uterus at hysterescopy. Although the upper reproductive tract seems to be a low microbial biomass site, our results suggest that the endometrial and FT microbiome is unique for each individual. In fact, samples obtained from the same individual from the endometrium and FT demonstrated more microbial similarity than for the same tissue (endometrium or FT) from two different women. Unraveling the female upper reproductive microbiome helps to understand the natural microenvironment in the tissues where the first processes of oocyte fertilization and embryo development and implantation are taking place and could therefore be used for improving in vitro fertilization and embryo culture conditions in treating infertile patients.