Chlamydia trachomatis is an important cause of sexually transmitted infections (STI) in Western countries. It is often asymptomatic, and thus, left untreated, and can have severe negative consequences, such as tubal infertility or adverse pregnancy outcomes. Other sexually transmitted microorganisms, such as Neisseria gonorrhoeae and Trichomonas vaginalis, as well as normal residents of the vaginal flora, such as genital mycoplasmas, also negatively impact human sexual and reproductive health. We evaluated the reliability of the Seegene Allplex STI Essential Assay for C. trachomatis detection using the real-time qPCR routinely used in our diagnostic laboratories as the gold standard. The Seegene assay displayed a sensitivity of 97.8% and a specificity of 98.9%. As this assay can also detect six other urogenital pathogens, we applied it to 404 samples from women who attended Lausanne University Maternity Hospital and obtained the following prevalence rates: 2.5% for C. trachomatis, 3.5% for Mycoplasma hominis, 6.3% for Ureaplasma urealyticum, and 27.7% for Ureaplasma parvum. Two samples were positive for Trichomonas vaginalis, and one sample was positive for Mycoplasma genitalium. Bacterial vaginosis was present in 4.5% of the cases and was strongly associated with M. hominis. Finally, we confirmed the association between C. trachomatis infection and pre-term birth (p = 0.03) but could not detect any association of this condition with other urogenital pathogens (Mycoplasma/Ureaplasma). In conclusion, given its high sensitivity and specificity for C. trachomatis DNA detection as well as its multiplex format, which simultaneously provides results for six other urogenital pathogens, the Seegene Allplex™ STI Essential Assay represents an appealing diagnostic tool in modern microbiology laboratories.