The aim of this paper is to demonstrate the importance of molecular diagnostics of bacteria and viruses in sexually transmitted infections (STIs) for the selection of appropriate therapy in their treatment. The materials used for the analysis include vaginal swabs taken from patients suspected of having STIs. The procedures for sampling, nucleic acid isolation, and pathogen detection through commercially available kits are described. The research results showed that the real-time PCR (RT-PCR) method is highly sensitive and specific for detecting the most common causes of STIs in women. Pathogens such as HPV, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, and Trichomonas vaginalis were successfully identified in all analyzed samples. Molecular methods, particularly RT-PCR, represent the gold standard in the diagnosis of sexually transmitted infections due to their high sensitivity, specificity, and speed. Their application enables early detection and targeted treatment of infections, which significantly improves treatment outcomes and reduces the risk of complications.