Improving biomass production with the utilization of low-cost substrate is a crucial approach to overcome the hindrance of high cost in developing large-scale microalgae production. The microalga Coelastrella sp. KKU-P1 was mixotrophically cultivated using unhydrolyzed molasses as a carbon source, with the key environmental conditions being varied in order to maximize biomass production. The batch cultivation in flasks achieved the highest biomass production of 3.81 g/L, under an initial pH 5.0, a substrate to inoculum ratio of 100:3, an initial total sugar concentration of 10 g/L, and a sodium nitrate concentration of 1.5 g/L with continuous light illumination at 23.7 W/m2. The photobioreactor cultivation results indicated that CO2 supplementation did not improve biomass production. An ambient concentration of CO2 was sufficient to promote the mixotrophic growth of the microalga as indicated by the highest biomass production of 4.28 g/L with 33.91% protein, 46.71% carbohydrate, and 15.10% lipid. The results of the biochemical composition analysis suggest that the microalgal biomass obtained is promising as a source of essential amino acids and pigments as well as saturated and monounsaturated fatty acids. This research highlights the potential for bioresource production via microalgal mixotrophic cultivation using untreated molasses as a low-cost raw material.