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Background: Sugars from sweet sorghum stalks can be used to produce ethanol and also to grow oleaginous yeasts. Instead of two separate processes, in this paper we propose a different route producing ethanol and microbial oil in two consecutive fermentation steps. Results: Three yeasts were compared in the first ethanol producing step. In the second step four different oleaginous yeasts were tested. Sweet sorghum juice was first clarified and concentrated. High gravity ethanol fermentation was carried out with concentrated juice with 23.7 g/100 mL of total sugars and without added nutrients. Total sugars were 2.5 times more than the original clarified juice. One yeast gave the best overall response over the two other tested; relative high ethanol productivity, 1.44 g ethanol/L·h−1, and 90% of sugar consumption. Aeration by flask agitation produced superior results than static flasks for all yeasts. Microbial oil production was done employing the residual liquid left after ethanol separation. The pooled residual liquid from the ethanol distillation contained 7.08 g/mL of total carbohydrates, rich in reducing sugars. Trichosporon oleaginosus and Lipomyces starkeyi produced higher dry biomass, total sugar consumption and oil productivity than the other two oleaginous yeasts tested; with values around 25 g/L, 80%, and 0.55 g oil/L·h−1 respectively. However, the biomass oil content in all yeasts was relatively low in the range of 14 to 16%. Conclusion: The two step process is viable and could be considered an integral part of a consolidated biorefinery from sweet sorghum.How to cite: Rolz C, de León R, Mendizábal de Montenegro AL. Co-production of ethanol and biodiesel from sweet sorghum juice in two consecutive fermentation steps. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.05.002.