Mycoplasma genitalium is a well-known cause of urethritis in men and has been associated with cervicitis, pelvic inflammatory disease, and adverse obstetric outcomes in women. In this cross-sectional study, we determined the current prevalence of M. genitalium infection and the rate of macrolide resistance in M. genitalium isolates, in patients visiting two large Dutch sexually transmitted infection (STI) clinics, to evaluate whether the recommendations in Dutch guidelines should be revised. In addition, risk factors for M. genitalium were identified. In total, 3225 patients were included. M. genitalium prevalence rates were 13.8% for all patients; 20.1% for men who have sex with men, 8.2% for men who have sex with women, and 12.6% for women. Macrolide resistance-associated mutations were detected in 66% of the patients infected with M. genitalium. Age, educational level, country of origin, number of sexual partners, HIV-positivity, infection with Neisseria gonorrhoeae, and urethral symptoms in men were independently associated with M. genitalium infection. In conclusion, we found very high prevalence rates and macrolide resistance rates of M. genitalium in patients visiting STI clinics.