Background and Objectives: Carotenoids are an important group of natural pigments with specific applications as colorants, food supplements, and additives; they are also used in medicine, cosmetics, and biotechnological purposes. In this study, carotenoid production by Rhodotorula mucilaginosa UIMC35 and its antifungal effect on Aspergillus fumigatus, Aspergillus flavus, and Mucor hiemalis, were investigated.   Methods: In this semi-experimental research, yeast was isolated from different natural sources and identified by macroscopic, microscopic, and biochemical tests. Then, yeast strain was identified using PCR technique. Carotenoids were extracted from the yeast strain by Davis method. After 48 hours, antifungal activity of the carotenoid, was determined against mold fungi according to growth inhibition percentage. The results were analyzed using estimated Kruskal-Wallis test.   Results: In this study, the isolated yeast strain was Rhodotorula mucilaginosa UIMC35. Growth inhibition percentage of Aspergillus fumigatus, Aspergillus flavus, and Mucor hiemalis, by Rhodotorula mucilaginosa UIMC35 carotenoids was determined to be 76.19, 66.67, and 21.05, respectively.   Conclusion: According to the results of this study, Rhodotorula mucilaginosa UIMC35 isolated from soil in Isfahan, could produce high potency carotenoids and the extracted pigment had a significant inhibitory effect against the studied mold fungi. Therefore, further researches can provide the possibility of using Rhodotorula yeast in various fields, such as pharmacy, food Industry, and cosmetics.