Glucosinolates, dormant defense compounds and secondary metabolites in plants of Brassicaceae family (broccoli, cabbage, mustard, kale, Brussels sprouts, etc.), become biologically active upon tissue damage in plant. Their activation triggers the “mustard oil bomb” mechanism, a potent chemical defense that influences both neighboring flora and herbivorous fauna. These compounds have become a major focus of scientific research due to their health-promoting properties, including cancer-preventive effects arising from bioactive breakdown products such as isothiocyanates, nitriles, and indoles. Glucosinolate synthesis consists of three basic steps that occur sequentially and involve numerous complex chain reactions. Conventional glucosinolate extraction from plant sources faces significant challenges such as low yields, seasonal variability, and high resource consumption. Therefore, microbial biosynthesis has emerged as a promising alternative offering sustainable, controllable, and scalable production platforms. Thanks to advances in metabolic engineering and synthetic biology, glucosinolate biosynthetic pathways have been reconstructed and optimized in microorganisms such as Escherichia coli and Saccharomyces cerevisiae. However, the seamless transfer of the complex synthesis cycle of these plant-based metabolites into microbial systems still poses significant challenges, such as ensuring high product titer, pathway stabilization, and efficient precursor supply. In this chapter, current strategies and progress in microbial glucosinolate production are discussed in detail, highlighting key enzymatic steps and host engineering approaches. In this way, a guiding framework for future studies on microbial production of glucosinolates is presented, and it is aimed to contribute to the transformation of research in this field into sustainable, efficient, and industrially applicable production technologies.