Microbial metabolomics is a study of a microbe’s metabolism and it reflects the actual physiological state of the microorganism. Its analysis focuses on intracellular and extracellular metabolites, signalling molecules and metabolic pathways. Moreover, metabolomics permits the identification of specific biomarker molecules associated with structural and metabolic states. Microbial metabolomics is a crucial component of the “omics” approaches applied in systems biology. Its approaches are targeted and untargeted depending on the research objectives and questions. Accessing the hidden diversity of natural products has become easier because of advancements in technologies and post-genomic developments. Currently, microbial metabolomics is commonly used in diagnostic applications, drug development, agriculture, bioremediation, environmental ecosystem and microbial metabolomics reconstructions. The chapter aims to provide an overview of microbe metabolomics and its challenges, as well as the development of new technologies, applications and future prospects for metabolomics.