Inexpensive carbon sources offering an alternative to glucose are searched for to reduce costs of docosahexaenoic acid production by microalgae. The use of waste glycerol seems substantiated and prospective in this case. The objective of this study was to determine the production yield of heterotrophic microalgae Schizochytrium sp. biomass and the efficiency of docosahexaenoic acid production in various types of cultures with waste glycerol. Cultivation conditions were optimized using the Plackett–Burman method and Response Surface Methodology. The highest technological performance was obtained in the fed-batch culture, where the concentration of Schizochytrium sp. biomass reached 103.44 ± 1.50 g/dm3, the lipid concentration in Schizochytrium sp. biomass was at 48.85 ± 0.81 g/dm3, and the docosahexaenoic acid concentration at 21.98 ± 0.36 g/dm3. The highest docosahexaenoic acid content, accounting for 61.76 ± 3.77% of total fatty acids, was determined in lipid bodies of the Schizochytrium sp. biomass produced in the batch culture, whereas the lowest one, accounting for 44.99 ± 2.12% of total fatty acids, in those of the biomass grown in the fed-batch culture.