Abstract: The chemical composition of bio-oil obtained from Arthrospira platensis biomass via hydrothermal liquefaction at 240–330°C has been studied with an elemental analysis and Fourier transform ultrahigh resolution mass spectrometry with ionic cyclotron resonance. An increase in temperature leads to an increased bio-oil yield, decreased oxygen, and an increase in the amount of carbon and nitrogen. The weighted Kendrick mass defect histogram showed for the first time that the main nitrogen-containing and oxygen-containing compounds are ON, O 2 N 3 , O 3 N 2 , ON 2 , N, and N 2 . The character of the change in their relative amount in bio-oil with a temperature change is also revealed. The Venn diagram shows the intersection of the sets of molecular formulas found in bio-oil samples obtained at different temperatures. The results may be used to optimize the hydrothermal liquefaction of microalgae and their subsequent processing into motor fuel.