Production of cellulosic ethanol from lignocellulosic biomass leads to the generation of a hemicellulosic hydrolysate during the feedstock pretreatment. This hydrolysate is rich in sugars, but also contains inhibitory compounds (mainly acetic acid and phenolic compounds) in concentrations that may be toxic to microbial growth. Currently, this side-stream of the cellulosic ethanol production process is processed as a waste, due to the lack of feasible alternatives for tackling the complexity of wood hemicellulosic hydrolysate. Thus, this work evaluated the ability of six microorganisms to metabolize the raw and detoxified hemicellulosic hydrolysate produced from Pinus taeda for the production of lactic acid, ethanol, xylitol, single-cell protein, lipids and carotenoids, with the aim of selecting a potential alternative for valorization of this side stream generated during the production of cellulosic ethanol contributing to the implementation of a sustainable advanced biorefinery. The tested microorganisms included a lactic acid bacterium, Bacillus coagulans; a probiotic bacterium, Lactobacillus salivarius; two oleaginous yeasts, Rhodosporidium toruloides and Saitoella coloradoensis; a thermotolerant yeast, Kluyveromyces marxianus; and a methylotrophic yeast, Hansenula polymorpha. L. salivarius, K. marxianus, and H. polymorpha showed promising ability to metabolize the partially detoxified hydrolysate (composed of (g/L): mannose, 29.27; glucose, 17.25; galactose, 6.18; xylose, 4.94; arabinose, 1.23; acetic acid, 7.99; formic acid, 4.86; levulinic acid, 4.04; 5-hydroxymethylfurfural, 0.74; total phenolic compounds, 0.40). On the other hand, the oleaginous yeasts and B. coagulans presented high sensitivity to the inhibitory compounds. L. salivarius produced lactic acid with high yield (1.1 g/g), which was limited by product inhibition. K. marxianus produced xylitol at 0.37 g/g xylose and ethanol at 0.19 g/g hexoses. Finally, H. polymorpha converted hexoses and acetic acid into single-cell protein with yield of 0.27 g/g. The production of lactic acid by L. salivarius proved to be a promising alternative for valorization of Pinus hemicellulosic hydrolysate in an ethanol biorefinery.