Introduction Population-based prevalence estimates of Mycoplasma genitalium (MG), Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in men and women in England are: 1.2% and 1.3%; 1.1% and 1.5%; and <0.1%, respectively. In sexual health clinics (SHCs), NG and CT are routinely tested for, whereas MG is not. Undiagnosed MG co-infection threatens and complicates empirical therapy of CT and NG, where azithromycin use may aid further spread of macrolide antimicrobial resistance (AMR). We assessed co-infection and macrolide AMR prevalence in symptomatic patients accessing three London SHCs. Methods Patients aged ≥16 years with symptoms of an STI provided samples: vulvovaginal swab (females), first void urine (men-who-have-sex-with-women (MSW) and men-who-have-sex-with-men (MSM)), pharyngeal and rectal swabs (MSM). Routine clinic CT/NG results were obtained and FTD Urethritis Plus kit used for MG detection. Resistance was determined using Sanger sequencing. Results Prevalence of NG only infection in females, MSW and MSM was 0.3% (95%CI 0–1.8), 3.5% (1.6–7.3) and 31.0% (21.4–42.5), respectively. MG only prevalence was 5.3% (3.3–8.4), 14.9% (10.4–21.0) and 11.3% (5.8–20.7), respectively. CT only prevalence was 5.6% (3.5–8.7), 15.5% (10.9–20.6) and 5.6% (2.2–13.6), respectively. MG-NG co-infection was in MSW only (0.6%, 0.1–3.2), representing 2.4% (0.4–12.3) of NG infections. CT-MG co-infection was in females and MSW (1.6%, 0.7–3.8% and 2.3%, 0.9–5.8, respectively), together representing 13.0% (7.0–23.0) of CT infections. CT-NG co-infection was in all groups (females: 0.3%, 0–1.8; MSW: 2.3%, 0.9–5.8; MSM 7.0%, 3.1–15.5). MG-NG-CT infection was found in females (0.7%, 0.2–2.4), representing 16.7% (4.7–44.8) of NG-CT infections. 64.9% (37/57) of MG samples sequenced were macrolide resistant (67.0% (21/31) from MSW). Conclusion With 13.0% and 2.4% of CT and NG infections respectively being co-infected with MG, and two-thirds MG infections displaying macrolide AMR, use of azithromycin for symptomatic CT/NG treatment in the absence of MG testing should be reconsidered.