Organic solid wastes are potential feedstocks for the production of liquid biofuels, which could be suitable alternatives to fossil fuels for the transport and heating sectors and for industrial use as well. By hydrothermal liquefaction (HTL), the wet biomass is partially transformed into a water-immiscible oil-like organic matter called bio-oil. In this study, three different mass spectrometric ionization techniques, namely, ESI, APCI, and APPI, in combination with a high-resolution FTICR mass analyzer were used in a comparative approach for the characterization of HTL bio-oil. In terms of the number of assigned molecular formulas, the three ionization techniques gave comparable results but with different distributions of the molecular classes. APPI, in particular, was demonstrated to be the ionization technique that best fits the actual elemental composition of the bio-oil sample. Our results, obtained by the integration of the three mass spectrometric ionization techniques, offer the opportunity to detect and identify by FTICR mass spectrometry the heteroaromatic compounds in bio-oil. Both aromatic molecules and nitrogen-containing species raise concern for the subsequent upgrading process of the bio-oil into a diesel-like fuel.