To develop multiplex PCRs (mPCRs) that allows simultaneous diagnosis of the infectious agents Chlamydia trachomatis, Toxoplasma gondii, HSV 1/2, and Cytomegalovirus (CMV). The study included patients with clinical suspicion of these agents, and clinical samples were blood, cerebrospinal fluid, urine, vaginal swabs, and amniotic fluid. After the extraction of DNA, this was used as a template in amplification by PCR of selected genes. The following conditions were tested: primer concentration, MgCl2 concentration, and annealing temperature. Three mPCRs were developed: multiplex I (CMV, HSV 1/2), multiplex II (CMV, HSV 1/2, T. gondii), and multiplex III (C. trachomatis, T. gondii, HSV 1/2, and CMV). The primer pairs used were shown to be specific for each infectious agent, and the specificity of mPCR assays was 100 %. Both the reactions of the monoplex PCR and mPCR produced a detection limit of 2 × 10(-5) to 6 × 10(-7) ng/μl of different DNAs. Upon conclusion, amplified products of expected size were obtained in 3 different reactions, and all the infectious agents were detected simultaneously in each mPCR. The concordant results of the study suggest that mPCR can be a powerful tool to improve the diagnostics of infectious diseases.