Hydrothermal liquefaction (HTL) is an attractive method for converting wet biomass into petroleum-like biocrude oil that can be refined to make petroleum products. This approach is advantageous for conversion of low-lipid algae, which are promising feedstocks for sustainable large-scale biofuel production. As with natural petroleum formation, the water in contact with the produced oil contains toxic compounds. The objectives of this research were to: (1) identify nitrogenous organic compounds (NOCs) in wastewater from HTL conversion of Spirulina; (2) characterize mammalian cell cytotoxicity of specific NOCs, NOC mixture, and the complete HTL wastewater (HTL-WW) matrix; and (3) investigate mitigation measures to reduce toxicity in HTL-WW. Liquid-liquid extraction and nitrogen-phosphorus detection was used in conjunction with gas chromatography-mass spectrometry (GC-MS), which detected hundreds of NOCs in HTL-WW. Reference materials for nine of the most prevalent NOCs were used to identify and quantify their concentrations in HTL-WW. Mammalian cell cytotoxicity of the nine NOCs was quantified using a Chinese hamster ovary (CHO) cell assay, and the descending rank order for cytotoxicity was 3-dimethylamino-phenol > 2,2,6,6-tetramethyl-4-piperidone > 2,6-dimethyl-3-pyridinol > 2-picoline > pyridine > 1-methyl-2-pyrrolidinone > σ-valerolactam > 2-pyrrolidinone > ε-caprolactam. The organic mixture extracted from HTL-WW expressed potent CHO cell cytotoxic activity, with a LC(50) at 7.5% of HTL-WW. Although the toxicity of HTL-WW was substantial, 30% of the toxicity was removed biologically by recycling HTL-WW back into algal cultivation. The remaining toxicity of HTL-WW was mostly eliminated by subsequent treatment with granular activated carbon.