The Gluconacetobacter xylinus species, belonging to a group of acetic bacteria, is capable of partial oxidation of glycerol. Dihydroxyacetone (DHA), being a product of that transformation, has been used in the food industry (as a sweetener), cosmetic industry (production of self-tanning creams) and in dermatology (treatment of leukoderma). The objective of the study reported herein was to determine values of the initial concentration of glycerol in the culture medium, active acidity of the medium and time of culture assuring the highest quantity of DHA produced, all three being optimal for the course of glycerol biotransformation to DHA. The biotransformation process was conducted with the use of G. xylinus cells immobilized in calcium alginate. The study demonstrated that in the culture medium with pH 5.0 and initial concentration of glycerol reaching 100 g/L, the concentration of DHA after 36 h of biotransformation accounted for 10.9 g/L. The highest content of the product in the culture media with active acidity of 7.0 or 8.0 and containing 100 g of glycerol/L at the beginning of the process, reached 10.5 g/L (after 36 h) and 10.7 g/L (after 34 h of the process), respectively. Elongating the biotransformation process to over 36 h resulted in a decrease in DHA concentration, which could have been due to its phosphorylation. Results obtained in this research demonstrated that the efficiency of glycerol biotransformation to DHA was determined, first of all, by the initial concentration of substrate and, to a slightly lesser extent, by acidity of the culture medium and process duration.