Lignocellulosic material is the most prominent carbon source to obtain profitable biotechnological processes, but compounds like furfural and acetic acid are highly toxic to yeasts. Nonetheless, research about the molecular mechanism of furfural and acetic acid toxicity is still scarce in yeasts like Scheffersomyces stipitis. Thus, this work aimed to identify the role of furfural and acetic acid on S. stipitis in bioenergetic and fermentation parameters. Here, we provide evidence that furfural and acetic acid caused a cell growth delay and extension of the lag phase. The mitochondrial membrane potential decreased in all treatments without differences among inhibitors or concentrations. Interestingly, the reactive oxygen species increased when the inhibitors concentrations were from 0.1 to 0.3% (v/v). The glycolytic flux was not significantly ( p>0.05) altered by acetic acid, but furfural caused different effects. The ethanol production decreased significantly (4.32 g/L in furfural and 5.06 g/L in acetic acid) compared to the control (26.3 g/L). At the same time, biomass was not significantly different in almost all treatments compared to the control. This study provides additional understanding of the effects of furfural and acetic acid at mitochondrial level in a pentose-fermenting yeast like S. stipitis.