Since the 1950s, the medical community has been faced with infectious diseases, which have brought significant public health and financial challenges. Currently, routine testing for the laboratory diagnosis for infectious agents is based on cell culture, serological, and molecular methods. However, cell culture-based methods are used mainly in research laboratories and are less sensitive methods when compared with serological and molecular methods. The diagnosis of infectious diseases has been revolutionized by the development of molecular techniques, mainly with the applications of polymerase chain reaction (PCR). The high sensitivity, specificity, and ease with which the PCR can be used to detect genetic sequences known have led to your wide application in science. A great number of qualitative and quantitative molecular assays are mostly based on what have been described such as real-time PCR, multiplex PCR, LAMP-PCR, and digital PCR. These assays could identify active infection by detecting infectious agents and nucleic acid in various clinical conditions including arboviruses, sexually transmitted infections, and bacterial infections. Further advancement of molecular technology is needed to improve the capacity to detect infectious agents in order to control the spread of infectious diseases and lead to appropriate actions which help to benefit patients and health-care workers themselves.