Abstract. Globally, there is growing interest in microalgae as production organisms. Microalgae contain lipids (oil), proteins and carbohydrates (sugars), and, especially marine algae have been used as food and feed for centuries. Recently, production cost reduction related to the supply of growth nutrients is necessary to make it profitable. Therefore, utilization of molasses, a byproduct of sugar production, as the natural carbon, macronutrients, and micronutrients sources can be helpful. The analysis showed that the content of sucrose, glucose, fructose, potassium, zinc, and magnesium was 68.4% w/w, 18.5% w/w, and 13.1% w/w, 5.5% w/w, 3.91 ppm, and 1,370 ppm respectively. This work aimed to determine the effect of molasses addition to the physio-chemical properties of multi-culture species of microalgae isolated from southern Java coastal region in Indonesia grown under mixotrophic culture. The cultivation in this work used medium which was selfformulated by the authors consisting of NaNO3 (5 mL/L), H3BO3 (1 mL/L), EDTA (1 mL/L), N2H2PO4 (5 mL/L), FeSO4 (1 mL/L), MgSO4 (1 mL/L), NaCl (1 mL/L), micronutrients (1 mL/L), vitamin B1 (1 mL/L), and vitamin B12 (1 mL/L) in 500 mL of water. The medium will be treated to have molasses concentration of 0. 05% v/v, 0.15% v/v, 0.25% v/v, 0.35% v/v, and 0.45% v/v.