Biotin (vitamin H) is one of the most fascinating cofactors involved in central pathways in pro- and eukaryotic cell metabolism. Since its original discovery in 1901, research has led to the discovery of the complete biotin biosynthesis pathways in many different microbes and much work has been done on the highly intriguing and complex biochemistry of biotin biosynthesis. While humans and animals require several hundred micrograms of biotin per day, most microbes, plants and fungi appear to be able to synthesize the cofactor themselves. Biotin is added to many food, feed and cosmetic products, creating a world market of 10-30 t/year. However, the majority of the biotin sold is synthesized in a chemical process. Since the chemical synthesis is linked with a high environmental burden, much effort has been put into the development of biotin-overproducing microbes. A summary of biotin biosynthesis and its biological role is presented; and current strategies for the improvement of microbial biotin production using modern biotechnological techniques are discussed.