The advent of advanced molecular technologies, particularly the widespread use of next-generation sequencing (NGS), has revolutionized the life sciences research landscape. NGS, characterized by high throughput, specificity, and sensitivity, offers a superior alternative to traditional detection methods, particularly in the context of viral pandemics and emerging infectious diseases. It has proven indispensable not only in identifying novel therapeutic targets but also in monitoring viral resistance. This review provides a comprehensive analysis of NGS applications in infectious diseases and pathogenic microorganisms, with an emphasis on its role in rapid pathogen detection, accurate identification, interspecies transmission tracking, and resistance profiling. Through the integration of these insights, the review offers a forward-looking perspective on emerging trends and challenges, highlighting the transformative impact of these technologies on the understanding and management of infectious diseases.