Hydrogen (H2) production from sugarcane press-mud, a waste obtained from the non-centrifugal sugarcane agroindustry, was assessed by coupling hydrolysis, fermentation, purification, and ethanol steam reforming (ESR). Two culture media were employed on three different sugarcane press-mud samples to produce bioethanol by fermentation using Saccharomyces cerevisiae at 30 °C. One culture medium was supplemented with nutrients and the other without supplementation. The supplementation did not have a significant effect over ethanol production (∼82.1 g L−1) after 70 h fermentation, but the concentration of the impurities was always lower under supplemented conditions. Among tested impurities, differences in 3-methyl-1-butanol showed the effect of the supplementation on the ESR over RhPt/CeO2-SiO2 catalyst at 700 °C, where the H2 yield decreased significantly in the presence of 3-methyl-1-butanol (p < 0.05). The spearman correlation coefficient showed that the H2 yield was correlated with the 3-methy-1-butanol content (RHO = −0.929) and carbon deposits (RHO = −0.964). Therefore, supplemented bioethanol could deliver 3.0 g H2 kg−1 sugarcane press-mud, which is almost twice that of the non-supplemented samples, likely due to the reduction of harmful impurities in the bioethanol. Additionally, supplemented conditions allowed for energy savings in the process and improved catalyst stability. This study provides insights into the effect of supplementing culture media to produce purer bioethanol samples, which further deliver higher H2 yields by ESR.