Production of bio-oil by hydrothermal liquefaction of biomass is a promising way to achieve several sustainable development goals: Renewable energy, proper waste treatment, reduction of greenhouse gas emissions, etc. For the wide application of bio-oil in industry and transport, it is important to study its molecular composition. However, the molecular composition of bio-oil is almost as complicated as that of natural petroleum, and despite the considerable amount of research, it is not fully understood. Here, we applied high-resolution mass spectrometry, collision induced dissociation, the isotope (H/D and 16O/18O) exchange approach, and other analytical techniques in order to obtain chemical information about individual molecules of bio-oil. We proved the presence of different nitrogen functionalities including quaternary nitrogen in the bio-oil. Analyzing MS/MS spectra of individual compounds, we proposed possible structures of individual molecules of bio-oil.