Vitamin C, C6H8O6, is a water-soluble vitamin that is widespread in nature. It is an essential nutrient involved in many biological processes in the living organisms: it enhances collagen biosynthesis, ensures the optimal functioning of enzymes and the immune system, has a major role in lipid and iron metabolism, and it enhances the biosynthesis of l-carnitine. Due to its antioxidant activity, vitamin C can neutralize the tissue-damaging effects of free radicals. Vitamin C is being related to the prevention of cancer and cardiovascular diseases. This review includes current information on the biosynthesis of ascorbic acid, as new methods are now challenging the traditional Reichstein process for vitamin C’s industrial-scale production. Different strains were analyzed in correlation with their ability to synthesize ascorbic acid, and several separation techniques were investigated for a more effective production of vitamin C.