OBJECTIVE To evaluate the association between Mycoplasma genitalium, Chlamydia trachomatis, and pelvic inflammatory disease (PID) METHODS: A case-control methodology was used. Swab eluates were processed using the QIAamp DNA mini kit. Polymerase chain reaction (PCR) for M. genitalium was carried out using a real time in-house 16S based assay. An endocervical swab was taken and tested for the presence of C. trachomatis (ligase chain reaction, Abbott Laboratories), and a high vaginal swab was taken and tested for the presence of Neisseria gonorrhoeae and bacterial vaginosis. RESULTS Of the PID cases 13% (6/45) had evidence of M. genitalium infection compared to none of the controls (0/37); 27% (12/45) of the cases had C. trachomatis infection compared to none of the controls; and 16% (7/45) of cases only had serological evidence of C. trachomatis infection compared to 5% (2/37) of controls. Cases were more likely to present with M. genitalium and/or C trachomatis than controls (p<0.001). CONCLUSIONS This study indicates that there may be an association between M. genitalium and PID, and that this relation is largely independent of C. trachomatis. Future studies need to investigate the pathological basis of the relation between M. genitalium and PID using samples from women with PID diagnosed using laparoscopy and endometrial biopsy. Little is known about the epidemiology of M. genitalium: large scale epidemiological investigations are needed to determine the prevalence, incidence, and factors associated with this emerging infection.