Numerous studies have demonstrated the efficacy of selenium compounds in preventing and treating lifestyle-related diseases such as cancer and cardiovascular disorders. The formulation of selenium-enriched supplements for humans and animals, particularly those containing selenium yeast, is highly advantageous. These products are rich in organic selenium derivatives, showing significantly higher bioavailability than inorganic forms of selenium. A particularly promising selenium analogue of sulphur-containing compounds is selenobiotin. The literature indicates that Phycomyces blakesleeanus and Escherichia coli strains can synthesise this compound. This research aimed to evaluate the effect of selenium supplementation on the biosynthesis of biotin and selenobiotin in Trichosporon cutaneum and Meyerozyma guilliermondii. The results have the potential to advance biotechnological approaches for the production of selenobiotin for various applications. A method based on affinity chromatography was used to quantify selenobiotin. The results confirmed that both yeast strains could synthesise selenobiotin in addition to biotin. In M. guilliermondii cells, selenobiotin accounted for up to 17.3% of the total biotin vitamer fraction. In comparison, in T. cutaneum cells, it accounted for up to 28.4% of the sum of biotin and its analogues. The highest levels of selenobiotin were observed in cells cultured with selenomethionine.