The aim of this study was to optimize biomass and docosahexaenoic acid (DHA) production by Schizochytrium sp. grown on waste glycerol as an organic carbon source. Parameters having a significant effect on biomass and DHA yields were screened using the fractional Plackett−Burman design and the response surface methodology (RSM). Schizochytrium sp. growth was most significantly influenced by crude glycerin concentration in the growth medium (150 g/dm3), process temperature (27 °C), oxygen in the bioreactor (49.99% v/v), and the concentration of peptone as a source of nitrogen (9.99 g/dm3). The process parameter values identified as optimal for producing high DHA concentrations in the biomass were as follows: glycerin concentration 149.99 g/dm3, temperature 26 °C, oxygen concentration 30% (v/v), and peptone concentration 2.21 g/dm3. The dry cell weight (DCW) obtained under actual laboratory conditions was 66.69 ± 0.66 g/dm3, i.e., 1.27% lower than the predicted value. The DHA concentration obtained in the actual culture was at 17.25 ± 0.33 g/dm3, which was 3.03% lower than the predicted value. The results obtained suggest that a two-step culture system should be employed, with the first phase focused on high production of Schizochytrium sp. biomass, and the second focused on increasing DHA concentration in the cells.