The revolutionary realm of molecular cloning, encompassing the creation of recombinant DNA molecules, has ignited a wave of progress within the life sciences. The advent of potent tools has facilitated the manipulation of DNA, resulting in an extraordinary surge in the versatility and breadth of applications in recombinant DNA technology. The once complex task of cloning genes has now been simplified, triggering a veritable explosion of insights into gene functionality. This has been achieved through the seamless fusion of multiple DNA fragments or the utilization of interchangeable gene cassettes, culminating in a state of unparalleled agility and expediency. In the 1970s, when restriction endonucleases, enzymes that cut DNA molecules selectively were discovered, molecular cloning technology has grown exponentially in application and intricacy, resulting in influential DNA manipulation tools. Recent decades have seen an explosion in our understanding of gene function due to the simplicity and efficiency of molecular cloning. It is expected that emerging technologies will offer superior potentials, such as stitching together multiple DNA fragments in under a few hours and transforming the resulting plasmids into bacteria, or the use of swappable genes, which can be easily moved between different paradigms, maximizing promptness and flexibility. It has been proved that cloning techniques provide a gold standard technique for polymicrobial infection, recombinant cytokines, antimicrobial peptides, epidemiology and gene therapy due to the limitations of culture-based methods. Due to molecular cloning technique, recombinant antigens are now being used to monitor patients against clinical infections. As a result of laboratory techniques that permit in vitro chemical synthesis of any DNA construct specified in silico, molecular cloning will likely undergo a paradigm shift in the coming future. As a result of these advances, DNA clones can be constructed faster and iteratively, which will speed up the growth of new vaccines, gene therapy vectors, and recombinant proteins. Here I present a detailed overview of the latest applications of molecular cloning techniques in medical microbiology.