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Background: Mycoplasmas (Mycoplasma spp. and Ureaplasma spp.) are sexually transmitted organisms found primarily in the human urogenital microbiota. These microorganisms are frequently associated with some diseases including non-gonococcal urethritis, vaginosis, pregnancy complications, prenatal infections, systemic infections and infertility. Objectives: The current studyaimedto evaluate the prevalence of Mycoplasma hominisandUreaplasmaspp. in routinegynecological care. Patients and Methods: A cross sectional study with simple random sampling was designed. A total of 6,810 patients with the age range of 11 to 80 years were evaluated, during a six-month period from January 2015 to June 2015 in Sao Paulo, Brazil. DNAs were extracted from cytological samples (ThinPrep™) to detect microorganisms by real-time polymerase chain reaction (qPCR). Results: The high prevalence of M. hominis (n = 79), Ureaplasma spp. (n = 2,026) and the co-colonization of both (n = 199) with P < 0.0001 in the samples were verified, mainly in sexually active aged females. The results also indicated a negative and significant correlation between co-colonization of Ureaplasma spp. and M. hominis, Ureaplasma spp., and age. Conclusions: In conclusion, the data showed that high rate of females harbor these microorganisms in the genital microbiota. This finding suggests the importance of routine gynecological tests, mainly in pregnant females, during the second and third trimesters, and in asymptomatic females under infertility clinical investigation. Furthermore, the application of molecular methods is suggested; since they provide a fast and accurate diagnosis of these microorganisms, but sensitivity of the tests should be considered in the clinical relevant concentration of microorganisms in the genital microbiota