Microbial biotransformation of waste streams into nutraceuticals appears to be a more viable approach to attaining sustainability. Thraustochytrids are leading producers of docosahexaenoic acid (DHA); an essential ω-3 fatty acid with several health benefits. A surge in identifying alternatives to glucose was observed for these heterotrophs in the past decade with little focus on their metabolic routes. In this study, Aurantiochytrium limacinum SR21 was evaluated for converting glycerol, acetic acid, and waste cooking oil into DHA. Compared to glucose, glycerol resulted in a similar biomass yield (9.53 g L-1) with higher biomass and DHA yield coefficients (YX/S & YP/S) i.e., 0.81 and 0.14 respectively. In contrast, acetic acid resulted in significantly lower biomass yields (5.07 g L-1) while lipid content was comparable. To explore synergistic effects, substrate combinations were tested, with glucose, glycerol, and acetic acid (GlcGlyAA) yielding the highest lipid (48.3 % DCW) and DHA content (45.6 % total fatty acids), approximately 1.2-fold higher than acetate alone. Conversely, WCO suppressed the effect of glycerol on lipid metabolism, resulting in lower DHA content (30.0 % total fatty acids). Further, untargeted metabolomics was performed for 12 combinations to understand the metabolic crosstalk between these substrates. The pentose phosphate pathway metabolites were enriched in glycerol, while fatty acid oxidation and the TCA cycle were more pronounced in WCO metabolism. Multivariate analysis exhibited that acetate combinations had a distinct metabolite profile enriched in γ-aminobutyric acid (GABA), suggesting its role in pathway regulation. GlcGlyAA showed significant accumulation of metabolites related to the pentose phosphate pathway and TCA cycle, yielding abundant acetyl-CoA and NADPH to support higher DHA. In conclusion, this study provides a roadmap and identifies key metabolic nodes that can be fine-tuned to enhance DHA yield.