The human body hosts trillions of diverse microbial species, along with their secreted metabolites and genes, collectively known as the human microbiome. Advances in bioinformatics, particularly culture-independent omics analyses, have enhanced our ability to study the microbiome and elucidate its functions. Understanding the roles of these microbes is crucial for comprehending human biology and developing novel therapeutics. The microbiome’s role in defending against invading pathogens is fundamental, starting with its critical contribution to the development and maturation of the immune system. This book chapter provides a comprehensive illustration of the microbiome’s role in defense, from mechanistic insights to therapeutic applications. We detail the interactions between the microbiota and the immune system, discussing various mechanisms and signaling pathways that underpin the antipathogenic activities of the microbiota, including colonization resistance, suppression of virulence factors, and the production of secreted antimicrobials. We also highlight unique host-microbiome-pathogen interactions across different body sites. We conclude by discussion the recent advances in the development of microbiome-based therapeutics to control infectious diseases. Besides, we analyze opportunities and challenges in the field, and explore the potential of machine learning tools in the discovery of novel antimicrobial molecules and the promising avenue of designing engineered live therapeutics.