Background: Crude extract obtained from the stem of Xylopia aromatica (Annonaceae, EB149) known as pimenta-de-macaco, a traditional Brazilian medicinal and edible plant, showed cytotoxicity against leukemia cell lines. Despite the initial findings, no information regarding its influence on behavioral phenotype (BP) has been previously reported. Objective: The objective was to assess the effect on EB149 on BP in male mice and to perform a bioguide-fractionation aimed at the verification of the cytotoxic potential against breast and prostate cancer cell lines. Materials and Methods: BP was assessed by observation in an open cage and subsequent analysis in an open field (OF) at 15, 30, 60, 120, and 180 min, after intraperitoneal administration of X. aromatica, in a two-stage experimental delineation. Results: EB149 impaired general activity, piloerection, defecation, breathing, auricular and corneal reflexes, tail squeeze, response to touch, hindquarter fall, surface-righting reflex, body tone, and grip reflex in the first stage. In the second stage, general activity, tail squeeze, touch response, and breathing were impaired, and a decrease in locomotion frequency in OF was also observed. All behavioral changes were recovered during the period of experiment in both stages. The LD50 of EB149 was 1.944 g/kg. Two fractions obtained from EB149 named FSIST, which contains sitosterol and stigmasterol as major compounds, and total alkaloid fraction, which contains total alkaloids, showed cytotoxicity against breast and prostate cancer cell lines. Conclusions: The traditional plant X. aromatica EB149 organic extract showed cytotoxicity against breast and prostate cancer cell lines and the recovery of behavioral impairment related to general activity, tail squeeze, touch response, breath, and locomotion frequency, causing no harm to male lab mice.