Alpha-linolenic acid (ALA), an important omega-3 polyunsaturated fatty acid, has important biological roles in cardiovascular protection, brain development, and anti-inflammatory responses. ALA traditionally comes from plant and marine sources, but microbial sources have also been used to provide an effective and sustainable substitute. This is because microorganisms have several advantages due to their rapid growth, their ability to use different substrates as carbon sources, and the ease with which genetic manipulations can be applied. This chapter provides a comprehensive review of microbial ALA formation by addressing bacterial fatty acid metabolism and the biosynthesis procedures that distinguish ALA from other omega-3 fatty acids. Various bacteria, yeasts, fungi, and microalgae involved in ALA synthesis are evaluated. The primary enzymes, metabolic processes, genetic control mechanisms, and the influence of environmental conditions on production efficiency of ALA biosynthesis are investigated. New developments in biotechnology such as systems biology techniques and gene editing tools are evaluated to enhance ALA production. The importance of microbial ALA production in nutrition and health is assessed, as well as its possible application in probiotics and functional foods. Finally, the importance of sustainability-based methods, alternative microbial production techniques, and synthetic biology for future research pathways is highlighted. In conclusion, microbial sources offer a creative and environmentally friendly way to meet the growing demand for omega-3 fatty acids worldwide.