Aiming to reduce energy expenditure, our research group recently proposed an innovative technology of falling film distillation assisted by a two-phase closed thermosyphon, patented as Destubcal. Second generation (2G) ethanol, produced from lignocellulosic materials is considered the biofuel with the highest potential to replace fossil derivatives. The objective of this study is to verify the effect of viscous components (such as glycerol and sugars) present in 2G-ethanol on the Destubcal distillation performance, in comparison to ethanol-water binary mixture. Two forms of heat supply in the steam chamber were evaluated: isothermal and temperature-profile, besides evaluating the influence of feed flow rate and evaporator temperature. When the thermosyphon’s steam chamber is operating in isothermal mode, temperatures of the bottom and the top of the distillation tube were higher with the 2G-ethanol mixture. This feedstock contains components with higher boiling temperature (as glucose and glycerol), which have influenced the temperature profiles. Unlike distillation with binary ethanol-water, lower evaporator temperatures favor 2G-ethanol distillation when the steam chamber is operating in temperature-profile mode. In all cases, the mass fraction of ethanol was favored in the 2G-ethanol distillation, indicating that the presence of components such as glucose and glycerol allows better recovery of the most volatile components in a falling film distillation process. The separation was similar with ethanol-water and 2G-ethanol mixture. However, the presence of components such as sugars and glycerol allowed better recovery of ethanol in the distillate. The imposition of a temperature-profile condition promoted higher ethanol mass fractions in the distillate, being the more suitable mode of operation.