Strychnos pseudoquina A. St.-Hil. (Loganiaceae), commonly known as "falsa-quina", "quina-branca" or “quina-do-cerrado”, is a native plant species found in the Brazilian Cerrado. Its stem bark has been traditionally used in folk medicine as a bitter tonic and for treating fever, malaria, liver, and stomach ailments. While there have been reports of the plant's beneficial properties, most species within the Strychnos genus are toxic and lack comprehensive data in the literature. Therefore, the objective of this study was to conduct a phytochemical investigation and evaluate the cytotoxic activity of the ethyl acetate fraction obtained from the stem bark of S. pseudoquina. The fraction exhibited significant inhibition of cell growth against all tested strains at a concentration of 50 µg mL-1, with inhibition values of 94.66% for HL60 (leukemia), 87.55% for HCT-116 (human colon carcinoma), 85.68% for PC3 (prostate), 72.31% for SNB-19 (astrocytoma), and 64.37% for L929 (mouse fibroblast). The phytochemical analysis resulted in the identification of two flavonoids, namely 3-O-methylquercetin and strychnobiflavone, along with two alkaloids, spermostrychnine and 12-hydroxy-11-methoxystrychnobrasiline, in the fraction. This is the first documented occurrence of these alkaloids in S. pseudoquina. © 2021 by the authors.