OBJECTIVES: A lactobacilli-dominated vaginal microbiome may protect against pelvic inflammatory disease (PID), but one dominated by Gardnerella species might increase susceptibility. Not all lactobacilli are equally protective. Recent research suggests that D(-) isomer lactic acid producing lactobacilli (Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri) may protect against infection with Chlamydia trachomatis, an important cause of PID. Lactobacillus iners , which produces L(+) isomer lactic acid, may be less protective. We investigated the microbiome in stored vaginal samples from participants who did or did not develop PID during the prevention of pelvic infection (POPI) chlamydia screening trial. METHODS: Long-read 16S rRNA gene nanopore sequencing was used on baseline vaginal samples (one per participant) from all 37 women who subsequently developed clinically diagnosed PID during 12-month follow-up, and 111 frequency matched controls who did not, matched on four possible risk factors for PID: age <20 versus ≥20, black ethnicity versus other ethnicity, chlamydia positive versus negative at baseline and ≥2 sexual partners in the previous year versus 0-1 partners. RESULTS: Samples from 106 women (median age 19 years, 40% black ethnicity, 22% chlamydia positive, 54% reporting multiple partners) were suitable for analysis. Three main taxonomic clusters were identified dominated by L. iners, L. crispatus and Gardnerella vaginalis. There was no association between a more diverse, G. vaginalis dominated microbiome and subsequent PID, although increased Shannon diversity was associated with black ethnicity (p=0.002) and bacterial vaginosis (diagnosed by Gram stain p<0.0001). Women who developed PID had similar relative abundance of protective D(-) isomer lactic acid producing lactobacilli to women without PID, but numbers of PID cases were small. CONCLUSIONS: In the first-ever community-based prospective study of PID, there was no clear association between the vaginal microbiome and subsequent development of PID. Future studies using serial samples may identify vaginal microbial communities that may predispose to PID.