The study aimed to determine the effectiveness of docosahexaenoic acid (DHA) production by Schizochytrium sp. biomass fed with waste glycerol depending on the concentration of extracellular polymeric substances (EPS) in the culture medium and medium aeration effectiveness. The microalgae from the genus Schizochytrium sp. were proved to be capable of producing EPS composed of glucose, galactose, mannose, fucose, and xylose. The highest EPS concentration, reaching 8.73 ± 0.09 g/dm3, was determined at the stationary growth phase. A high EPS concentration caused culture medium viscosity to increase, contributing to diminished oxygen availability for cells, lower culture effectiveness, and reduced waste glycerol conversion to DHA. The Schizochytrium sp. culture variant found optimal in terms of the obtained technological effects and operating costs was performed at the volumetric oxygen mass transfer coefficient of kLa = 600 1/h, which enabled obtaining dry cell weight (DCW) of 147.89 ± 4.77 g/dm3, lipid concentration of 69.44 ± 0.76 g/dm3, and DHA concentration in the biomass reaching 29.44 ± 0.36 g/dm3. The effectiveness of waste glycerol consumption in this variant reached 3.76 ± 0.31 g/dm3·h and 3.16 ± 0.22 g/gDCW.