Biomass pre-treatment is a key step in achieving the economic competitiveness of biomass conversion. In the present work, an imidazole pre-treatment process was performed and evaluated using wheat straw and eucalyptus residues as model feedstocks for agriculture and forest-origin biomasses, respectively. Results showed that imidazole is an efficient pre-treatment agent; however, better results were obtained for wheat straw due to the recalcitrant behavior of eucalyptus residues. The temperature had a stronger effect than time on wheat straw pre-treatment but at 160 °C and 4 h, similar results were obtained for cellulose and hemicellulose content from both biomasses (ca. 54% and 24%, respectively). Lignin content in the pre-treated solid was higher for eucalyptus residues (16% vs. 4%), as expected. Enzymatic hydrolysis, applied to both biomasses after different pre-treatments, revealed that results improved with increasing temperature/time for wheat straw. However, these conditions had no influence on the results for eucalyptus residues, with very low glucan to glucose enzymatic hydrolysis yield (93% for wheat straw vs. 40% for eucalyptus residues). Imidazole can therefore be considered as a suitable solvent for herbaceous biomass pre-treatment.