The catalytic activity of high-loaded Ni-based catalysts for beech wood fast-pyrolysis bio-oil hydrotreatment is compared to Ru/C. The influence of promoter, temperature, reaction time, and consecutive upgrading is investigated. The catalytic activity is addressed in terms of elemental composition, pH value, H₂ consumption, and water content, while the selectivity is based on the GC-MS/FID results. The catalysts showed similar deoxygenation activity, while the highest hydrogenation activity and the highest upgraded oil yields were obtained with Ni-based catalysts. The elemental composition of upgraded oils was comparable for 2 and 4 h of reaction, and the temperature showed a positive effect for reactions with Ni–Cr and Ru/C. Ni–Cr showed superior activity for the conversion of organic acids, sugars and ketones, being selected for the 2-step upgrading reaction. The highest activity correlates to the strength of the acid sites promoted by Cr₂O₃. Consecutive upgrading reduced the content of oxygen by 64.8% and the water content by 90%, whereas the higher heating value increased by 90.1%. While more than 96% of the organic acid content was converted, the discrepancy of aromatic compounds quantified by ¹H-NMR and GC-MS/FID may indicate polymerization of aromatics taking place during the second upgrading step.