| Summary: | <i>Aspidosperma pyrifolium</i> is used in traditional medicine to treat inflammatory disorders. The aim of the study was to perform phytochemical characterization and evaluate the anti-inflammatory, anti-nociceptive and acute toxicity effects of the total alkaloid fraction (TAF-Ap) from stem barks. Two monoterpenic indole alkaloids were isolated by high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and the structural elucidation was performed using 1D and 2D NMR analysis. As for toxicity, no animals died at 50 mg kg<sup>−1</sup> and this concentration presented mild sedation and forced breathing within the first 24 h. The lethal dose capable of killing 50% of the animals (LD<sub>50</sub>) was estimated to be 160 mg kg<sup>−1</sup>. In the pharmacological tests, the models used were 1% carrageenan-induced paw edema and peritonitis, 1% formalin-induced nociception and 1% acetic acid-induced abdominal writhing in Swiss mice. The study made it possible to isolate 15-methoxyaspidospermine and 15-methoxypyrifolidine, corroborating the results of pharmacological assays, which showed anti-inflammatory and analgesic potential, especially at 30 mg kg<sup>−1</sup> (<i>p</i> < 0.001). Thus, the species was shown to be a promising source of active substances, with special attention paid to its toxicological potential.
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