Background Deproteinized potato wastewater and glycerol are two by-products which are difficult to dispose. The objective of this study was to determine the ability of Rhodotorula glutinis to use glycerol and nitrogen compounds present in deproteinized potato wastewater and to evaluate the ability of simultaneous biodegradation of potato wastewater and glycerol via microbiological methods. Results It has been found that R. glutinis used glycerol and potato wastewater as a source of carbon and nitrogen, respectively. The highest degree of glycerol content (70.6%) reduction was found after cultivation of the investigated strain using a medium with 5% glycerol. In this medium, a significant reduction in the total protein content, estimated at 61%, was observed. The process of 72 h cultivation of yeast in a medium containing potato wastewater and 5% glycerol reduced the chemical oxygen demand (COD) more than 77%. Supplementation of media with high doses of glycerol (i.e. 20 and 25%) led to decreased metabolic activity in the yeast strain tested. Conclusion It has been found that there is a possibility of simultaneous biodegradation of potato wastewater and glycerol during the cultivation of R. glutinis.