Background: There is an ongoing debate regarding possible cost and benefits, but also harm of universal screening for the emerging sexually transmitted pathogen Mycoplasma genitalium. Methods: From the initial pool of 8665 samples that were tested, a subset of Chlamydia trachomatis-positive and randomly selected C. trachomatis-negative cervical swabs were further interrogated for M. genitalium by real-time polymerase chain reaction, using a 224 bp long fragment of the glyceraldehyde-3-phosphate dehydrogenase gene. Results: M. genitalium was detected in 4.8% of C. trachomatis-positive samples and none of C. trachomatis-negative samples. Accordingly, a significant association was shown between M. genitalium and C. trachomatis (P < 0.01), but also between M. genitalium and Mycoplasma hominis infection (P < 0.01). Conclusions: Based on the results, routine screening is recommended only for women with one or more identified risk factors. Moreover, younger age does not represent an appropriate inclusion/exclusion criterion for M. genitalium testing in the low-risk female population.