Various harmful microbes, pests, and insects are major sources of infection in plants. A variety of microorganisms, such as bacteria, fungi, and actinobacteria, which show the potential of defense mechanisms against plant and soil-borne pathogens, are generally recognized globally as biocontrol agents (BCAs). A variety of BCAs, such as Bacillus cereus, Pseudomonas putida, Trichoderma, Bacillus amyloliquefaciens, and other rhizospheric microbes, have been investigated and registered to serve their antagonistic properties for protecting plants such as small millet, tomato, sunflower, rice, and potato. In addition, BCAs also regulate plant nutrition, which may increase the concentration of various important metabolites such as flavonoids, alkaloids, terpenes, saponins, amino acids, sugars, lipids, and organic acids. Therefore, the analysis of these metabolites can be performed by an effective and advanced area called plant metabolomics, which provides a great understanding of the metabolic characteristics of the biological system. The function and role of small metabolites can be better recognized by metabolomics tools, namely, LC-MS, GC-MS, and other omics technologies. These technologies play a major role in crop improvements, such as increasing phytohormones and aiding plants in developing complex defense systems against various plant pathogens, boosting plant survival, and building metabolisms for biotic stress. Hence, there is a need to develop novel microbial BCAs that are more effective and eco-friendly for sustainable agriculture and could replace the use of hazardous compounds. This chapter highlights the importance of plant metabolomics and its application to biotic stress by BCAs and their impact on various pathogens.