Background: Certain high-risk (hr) types of human papillomavirus (HPV) can cause cervical cancer in women and penile cancer in men. Hr-HPV can also cause cancers of the oropharynx and anus in both sexes. In the anal and cervical region, a contribution of co-infections with Ureaplasma spp. on the persistence of the hr-HPV infection by a profound inflammatory state is suggested. Here, we investigated if non-HPV sexually transmitted infections are associated with oropharyngeal carcinoma (OPC). Materials and Methods: In this case-control study, a brush test directly from the tumor surface of OPC patients (study group) and from the oropharynx of healthy volunteers (control group), both groups matching in age and sex, was performed. HPV subtypes were detected using a commercially available test kit. For non-HPV sexually transmitted infections (Ureaplasma spp., Chlamydia trachomatis, Mycoplasma hominis, and Mycoplasma genitalium), a multiplex nucleic acid amplification approach was performed. Results: In the study group, 96 patients (23 female/73 male), with histologically confirmed OPC and in the control group 112 patients (19 female/93 male), were included. Oropharyngeal hr-HPV-positivity was detected in 68% (65/96 patients) of the study group and 1.8% (2/112 patients) of the control group (p < 0.001). In three patients in the study group, Ureaplasma spp. was detected, whereas no patient was Ureaplasma spp. positive in the control group (p = 0.097). Chlamydia trachomatis, Mycoplasma hominis, and Mycoplasma genitalium were negative in both groups. Conclusion: Based on the current study, the prevalence of oropharyngeal Ureaplasma spp. among patients with OPC is low and does not support a role in oropharyngeal cancer. However, the detection of the pathogen only among OPC patients but not in the healthy individuals might indicate a potential role and needs further elucidation.