This study examined the prevalence and factors associated with Mycoplasma genitalium (MG) infection among HIV-positive women and the association between MG and vaginal HIV-1 RNA shedding. HIV-positive women attending an outpatient clinic in New Orleans, Louisiana, USA, from 2002 to 2005 were examined for a battery of sexually transmitted infections (STIs) and underwent a behavioural survey. A selected subset had a measurement of vaginal shedding analysed. Of the 324 HIV-positive women, 32 (9.9%) were infected with MG. HIV-positive women with MG were more likely to be co-infected with Neisseria gonorrhoeae and Chlamydia trachomatis and to have had ≥1 male sexual partners in the last month. In the subset (n = 164), no differences were found in the presence of detectable vaginal HIV-1 RNA between women infected and not infected with MG (30.8% versus 34.8% shedding; P = 0.69). While MG was a common co-STI in this sample of HIV-positive women, it was not associated with vaginal HIV shedding.