Microbial Secondary Metabolites (SMs) are low molecular weight compounds produced during secondary metabolism. The majority are produced by specific groups of microorganisms under environmental stress conditions, including; nutrient deficiencies, pH, temperature, and metal ions concentrations. Many reported SMs are categorized as antibiotics, pigments, toxins, pheromones, enzyme inhibitors, antitumor agents, immunomodulators, receptor antagonists and agonists, animal and plant growth promoters, and pesticides. Their synthesis is regulated by a group of genes residing on the chromosomal DNA/plasmid DNA known as Biosynthetic Gene Clusters (BGCs). Moreover, their metabolic pathways are complex and controlled by many regulating factors, thus needing to be studied from an enzymatic, regulatory, and differentiation perspective. Due to their structural diversity, SMs have a wide range of biotechnological applications in the agricultural, health, industrial, and environmental sectors. Hence, they have a great potential to positively impact health, nutrition, and the economy on a global scale. The main goal of this Research Topic is to highlight current research exploring microbial SMs and their potential future applications in the agricultural, pharmaceutical, industrial, and environmental sectors. Microbes have proved to be a reliable source of SM production, with high structural diversity and product yields. However, there are several challenges to reliable SM production, including; the regulation of cultural conditions, development of industrial strains, and product yield. Such challenges can be mitigated to some extent through exploring non-conventional microbial sources, chemical and biological modifications, metagenomics, and genome mining approaches. Additionally, genetic engineering techniques can improve microbial strain characteristics, product yield, and generate novel molecules. Furthermore, microbial co-culture methods have been praised for increasing the yields and improving the structural diversity of the product, as well as advances in computational strategies enabling the identification of BGCs in genome sequences and the prediction of chemical product structures. Nevertheless, there remains significant potential to identify novel and industrially important SMs.