Background: Infectious diseases are caused by pathogenic microorganisms that spread directly or indirectly from one person to another. They are the main global cause of morbidity and mortality, seriously affecting public health and socioeconomic stability.They can be caused by a wide range of pathogens with similar clinical manifestations. Selection of appropriate techniques for rapid pathogen identification is critical for disease diagnosis and public health management. Real Time-PCR is a molecular method of proving nucleic acid directly in the patient's material, in which multiplication and quantification of pathogen DNA takes place simultaneously. It has high sensitivity, specificity, and an important role in the early and rapid detection of pathogens. Methods and Objectives: In the detection of pathogens of infectious diseases, a molecular method, Real Time PCR, was used. The aim of the work is to show the advantages of using molecular diagnostics in the fight against infectious diseases, to consider its applicability in controlling infectious diseases, reducing their spread and rapid public health response to serious public health events. Results: In the period from June 2022 until June 2024, a team of two two laboratory technicians, one medical microbiology specialist and one biologist are employed at the Department for Molecular Diagnostics at Institute of Public Health of Šabac, processed and tested 40,595 samples for the needs of patients in the Mačva and Kolubara districts. The tested samples painted an epidemiological picture of the district. Conclusion: The unexpected emergence of infectious diseases in the last decade has been rapidly detected using molecular methods. Information obtained from RT-PCR testing provided early insights that guided the public health response. In an environment with limited resources and a wide range of possible etiologies, molecular technologies offer an efficient solution for the diagnosis of infectious diseases, because they are fast, flexible and agile. It makes it possible to cover a large territory with few resources. Health systems that routinely use molecular diagnostics will achieve economies of scale, maximize limited expertise, and respond rapidly to emerging threats.