Objectives <p>This study aims to assess the prevalence of Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), and Neisseria gonorrhoeae (NG) among outpatients of reproductive medicine center in Chongqing, China.</p> Methods <p>This retrospective study included patients who attended the Center for Reproductive Medicine of Women and Children’s Hospital of Chongqing Medical University during the period from 2018 to 2022. Semen or cervical swab samples were collected from patients and subsequently analyzed for the presence of CT, MG, and NG pathogens using real-time polymerase chain reaction (RT-PCR).</p> Results <p>This study included 111,222 patients (mean age: 33.28 ± 5.49 years, range: 19–76 years), including 53,103 males and 58,119 females. The prevalence of CT positivity was significantly lower in males than females (1.93% vs. 2.57%, <i>p</i> &lt; 0.001), whereas MG positivity was higher in males (3.57% vs. 2.04%, <i>p</i> &lt; 0.001). The prevalence of NG positivity was comparable between genders (0.12% vs. 0.11%, <i>p</i> = 0.612). Notably, the prevalence of CT decreased with age in both male and female patients, while the prevalence of MG and NG remained relatively stable across age groups. Among all the infected patients, 95.1% of male patients and 95.8% of female patients were infected with only one of the pathogens, while the predominant mixed infection is co-infection with CT and MG.</p> Conclusions <p>In Chongqing infertility patients, CT and MG infections exceeded NG, with single infections predominating. This high prevalence necessitates integrated sexually transmitted infection screening in infertility care.</p>