Riboflavin (vitamin B2) is an important nutrient used as a precursor for the synthesis of flavin nucleotides, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), which are used as coenzymes in many enzymatic reactions, mostly oxidoreductive. Riboflavin is widely used in food, agriculture, and medicine. Riboflavin is synthesized by many bacteria and by all fungi and plants, but humans and other animals need to acquire sufficient vitamins with their diet or through supplements to maintain optimal health. The annual market for this vitamin is estimated at US $451.57 million in 2024. Traditionally, riboflavin has been produced chemically, but microbial synthesis is gaining traction due to lower production costs, reduced waste, and decreased energy consumption. Despite these advantages, the genetic stability of microbial overproducers remains a challenge as most yeasts and filamentous fungi naturally produce riboflavin in amounts sufficient only for their metabolic needs. Yeasts and filamentous fungi mostly produce small amounts of riboflavin, which corresponds to their needs. However, there is a group of yeasts and filamentous fungi, known as flavinogenic organisms, which are able to produce larger amounts of riboflavin. Flavinogenic yeasts overproduce riboflavin under iron starvation, and flavinogenic fungi do it during sporulation in the stationary growth phase. Some mutants of the flavinogenic fungus Ashbya gossypii and the yeast Candida famata belong to the most effective riboflavin overproducers known. This review discusses the development and application of molecular tools, including transformation systems, selectable markers, promoters, and clustered regularly interspaced palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) technology, to enhance riboflavin production in these organisms. The successful genetic engineering of A. gossypii and C. famata highlights the potential for more efficient and sustainable industrial riboflavin production.