Objective: To describe the microbiome of the vagina and fallopian tubes, and its relation with hydrosalpinx. Design: Prospective case-control study. Population and Settings: Women who underwent salpingectomy for hydrosalpinx or other indications at our medical center . Main Outcome Measures: Samples obtained during surgery and were subjected to 16S rRNA amplicon sequencing, and analyses of alpha diversity and beta diversity meseaures were compared between sites and groups. Differential abundances of bacteria associated with vaginal dysbiosis was compared between cases and controls. Results: Nine women with hydrosalipnx, and 23 women without hydrosalpinx were included in the study. The mean age of studied women was 41 (range: 29-54), and most (89%) were premenopausal. After in silico decontamination, only 30% of control fallopian tubes samples, and 10% of case fallopian tubes samples, had evidence of bacterial presence. The vaginal microbiota of control patients showed greater abundance of lactobacilli whereas the vaginal microbiome of case patients contained relatively more bacterial vaginosis associated bacteria such as Prevotella, Gardenrella and Atopobium. A significant difference was found in alpha and beta diversity between the vaginal and FT microbiomes in control patients, as fallopian tubes samples were more diverse. We found that women with hydrosalpinx had a more “dysbiotic” vaginal microbiome, and in women without hydrosalpinx, microbial composition within the vagina and FT differed, possibly representing two distinct ecological environments. Conclusion: women undergoing salpingectomy for various reasons harbored bacteria within their FT, women with hydrosalpinx generally did not. This suggests that even though infection may be an underlying cause of hydrosalpinx, bacteria may not be present by the time patients require surgery.