Euglena gracilis’s mixotrophic metabolism offers biotechnological potential. This study investigated how glucose, sodium acetate, ethanol, and propanetriol regulate its growth, photosynthesis, and paramylon production. All carbon sources boosted paramylon yield versus photoautotrophic controls. Ethanol and glucose were both highly effective, supporting the highest biomass accumulation (5.71 and 4.42-fold increases, respectively) and paramylon content without a significant difference between them. Ethanol supplementation enhanced chlorophyll b via coupled TCA cycle/glyoxylate shunt activity, while glucose showed the strongest tendency for high paramylon and the highest carotenoid content (13.36-fold higher). Sodium acetate triggered alkaline stress (pH 8.5), suppressing pigments and inducing spherical cells. Propanetriol reduced biomass but enhanced PSII efficiency (Fv/Fm). These results demonstrate carbon source-driven metabolic partitioning: ethanol and glucose both excel in promoting growth and storage, while additionally directing carbon toward chlorophyll b or carotenoids, respectively. These findings enable targeted bioprocess optimization: selection between ethanol or glucose can be based on the value of co-products, advancing E. gracilis as a sustainable cell factory.