Menthol, a monoterpene alcohol widely used in pharmaceutical, cosmetic, and food industries, is traditionally derived from plant extraction or chemical synthesis. However, these methods face challenges such as resource limitations, environmental concerns, and high production costs. Microbial biosynthesis has emerged as a sustainable and scalable alternative, leveraging metabolic engineering and synthetic biology to enhance menthol production. This chapter elucidates the natural biosynthesis of menthol in Mentha species, detailing key enzymatic steps and metabolic intermediates. It then examines microbial platforms for menthol production, including bacteria (Escherichia coli, Corynebacterium glutamicum), yeast (Saccharomyces cerevisiae, Yarrowia lipolytica), and fungi (Aspergillus, penicillium), highlighting their advantages, limitations, and genetic optimization strategies. Synthetic biology approaches such as precursor pathway engineering, enzyme optimization, and pathway flux balancing are discussed to improve yield and efficiency. The chapter concludes with future perspectives on enhancing scalability and industrial viability through adaptive laboratory evolution, nontraditional microbial hosts, fermentation strategies, and bioprocess optimization—including substrate selection, fermentation modes, process conditions, and downstream processing. By integrating advanced biotechnological innovations, microbial menthol biosynthesis offers a promising path toward sustainable, cost-effective, and scalable production.