The aim of this paper is to describe the prevalence of Mycoplasma genitalium and Trichomonas vaginalis in patients who visited general practitioners in the Netherlands. Additionally, we describe the prevalence of M. genitalium resistance to azithromycin and moxifloxacin. We used data from 7,411 consecutive female patients who were screened for Chlamydia trachomatis, Neisseria gonorrhoeae, M. genitalium, and T. vaginalis and data from 5,732 consecutive male patients screened for C. trachomatis, N. gonorrhoeae, and M. genitalium. The prevalence of M. genitalium and T. vaginalis in female patients was 6.7% (95% CI: 6.2 to 7.4) and 1.9% (95%CI: 1.6 to 2.2%), respectively. M. genitalium prevalence in male patients was 3.7% (3.3 to 4.3). M. genitalium co-occurred with C. trachomatis in 1.4% (0.3 to 0.6%) of female and in 0.7% (0.5 to 0.9) of male patients. Macrolide resistance gene mutations and fluoroquinolone resistance gene mutations were detected in 73.8% and 9.9%, respectively. We concluded that M.genitalium is relatively infrequently found in a large general practitioner population in the Netherlands. It can co-occur with C. trachomatis, and is often resistant to azithromycin. Therefore, when treating sexually transmitted infections, these prevalence and resistance data should be taken into account.