Control of carotenoids production from the yeast Rhodotorula mucilaginosa AY-01, based on the results of 28S rDNA sequence (D1/D2 region) analysis and homology search, was evaluated under different carbon and nitrogen sources to establish the optimal condition. Herein, we report that the control of production facilitates the extraction of intracellular carotenoids from yeast R. mucilaginosa AY-01. The use of methanol only for carotenoids production was found to be the best approach when compared with other enzymatical, chemical or physical processes. Glucose partially purified from carotenoids using an immobilized metal affinity chromatography (IMAC) was identified as the major monosaccharide with minor unidentified sugars by high performance anion-exchange chromatography (HPAEC) analysis. The glucosidal carotenoids extracted from the yeast R. mucilaginosa AY-01 showed highly significant antioxidant activity. In addition, a significant antibacterial activity of glucosidal carotenoids toward antibiotic resistant bacteria (ARB) was elucidated with potential biological applications in various food, cosmetic and/or hygiene supplements industries. Collectively, our study describes the production and characterization of a red pigment with antioxidant and antibacterial activities from the yeast R. mucilaginosa AY-01. (C) 2016 Elsevier Ltd. All rights reserved.