Abstract <p>Riboflavin (vitamin B₂) is an essential compound for the nutrition of living organisms, serving as a precursor of coenzymes flavin mononucleotide and flavin adenine dinucleotide, which are involved in numerous enzymatic reactions mainly in oxidative metabolism. The yeast <i>Candida famata</i> is a natural riboflavin-producing species, which are able to oversynthesis riboflavin under conditions of iron starvation. The aim of this study was to construct recombinant <i>C. famata</i> strains with increased riboflavin production by the simultaneous overexpression of the three genes: <i>RFE1</i>, <i>GND1</i>, and <i>RIB6</i>, which encode riboflavin excretase, 6-phosphogluconate dehydrogenase and 3,4-dihydroxy-2-butanone-4-phosphate synthase, respectively. The expression of various combinations of two genes, as well as the co-expression of all three genes, resulted in increased riboflavin production in <i>C. famata</i> VKM Y-9 in different media. The strains engineered for the co-overexpression of all three genes exhibited up to a 3.3-fold increase in riboflavin production by the fifth day of cultivation in cheese whey, compared to the parental strain.</p>