Abstract Bioethanol is one of the renewable alternative energies and can be used as a substitute for fossil fuels. Bioethanol is produced from a fermentation process, which is assisted by microbial yeast groups (ethanologenic yeast). Yeast Pichia kudriavzevii is capable of converting both carbon source pentose and hexose sugars to ethanol. In the previous study, P. kudriavzevii ethanol stress - tolerant mutant strains T5 was successfully constructed via directed mutagenesis thus suggesting their potential as fermentation agent. One strategy to increase ethanol production is by applying co-cultivation techniques. Thus, this study aimed to examine the ability of sugar substrate utilization by ethanologenic yeast S. cerevisiae and P. kudriavzeviiat various inoculum ratio. The results of the study indicated that no antagonist interaction detected between S. cerevisiae with all of P. kudriavzevii, both mutant and wild type strains. Based on sugar consumption analysis, both yeast isolates could be used in ethanol production simultaneously to maximize mixed substrate utilization. Our study revealed that the best sugar consumption found on co-cultivation of S. cerevisiae with wild type P. kudriavzevii T (wt) in 1: 1 inoculum ratio.