Background Rectal sexually transmitted infections (STI) are common in men having sex with men (MSM). Mycoplasma genitalium is increasingly being reported in this localization, but due to frequent lack of symptoms at this site, clinical significance is still unclear. Rectal prevalence of Mycoplasma hominis and Ureaplasma species is not well studied so far. We aimed to investigate the prevalence and antibiotic sensitivity of rectal Mollicutes in our HIV-cohort. Methods In 227 MSM presenting for annual STI-screening, 317 anorectal swabs were collected from January 2017 to December 2018. PCR was performed for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, M. genitalium and also culture for M. hominis and Ureaplasma spec. Results Prevalence for M. genitalium, M. hominis, Ureaplasma spec., C. trachomatis and N. gonorrhoeae was 8.2%, 7.3%, 12.0%, 5.1% and 1.9%, respectively. Patients were asymptomatic with few exceptions. Seroprevalence of syphilis in 227 MSM was 41.9%. In 20 strains of M. genitalium, resistance-associated mutations to macrolides and quinolones were found in 60% and 30%, respectively; in five strains (25%) to both. M. hominis and Ureaplasma spec. frequently occurred combined, mostly in significant quantity consistent with infection. M. hominis and Ureaplasma spec. regularly showed sensitivity to tetracycline. Conclusion At screening, rectal colonization with Mollicutes was common in our patients, but rarely caused symptoms. Due to rising antibiotic resistance of M. genitalium against quinolones, therapeutic options are increasingly limited. Treatment should be guided by antibiotic resistance testing including quinolones. In persisting anorectal symptoms, M. hominis and Ureaplasma spec. should also be taken into account.