This study employed culture and polymerase chain reaction (PCR) to examine the prevalence of Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma fermentans, Mycoplasma penetrans and Mycoplasma pirum in 210 HIV/AIDS patients, 455 sexually transmitted infection (STI) clinic attendees and 245 healthy volunteers from first-void urine specimens for men and endocervical swabs for women. U. urealyticum and M. hominis were detected in 107 (51.0%) and 69 (32.9%) patients in the HIV/AIDS group. At least one of the other four organisms was detected in 34 (16.2%) HIV/AIDS patients, 29 (6.4%) STI clinic attendees and six (2.5%) healthy volunteers. This study showed that U. urealyticum, M. hominis and M. fermentans were significantly more prevalent in HIV/AIDS patients, as were other mycoplasmas. Our results suggest a possible role for co-infection.