Considered safe because being from natural origin, plant products can exhibit toxic effects during their use. To ensure safe use of Annona senegalensis Pers. (Annonaceae), plant widely used for its therapeutic potential, the phytochemical and safety profiles of its root wood were investigated. Standard tube characterization tests were used to highlight phytochemical groups of root wood of the plant. Acute and subacute toxicity tests were carried out in Medical Research Institute (NMRI) mice following the Organization for Economic Cooperation and Development (OECD) test guidelines. The phytochemical screening showed the presence of sterols and triterpens, polyphenols, reducing compounds, and flavonoids in root wood of Annona senegalensis (A. senegalensis). In toxicological studies, the results indicate that the aqueous extract has a low acute toxicity with an LD50 greater than 5000 mg/kg b.w. The results of subacute toxicity study indicate that the mice receiving 300 mg/kg b.w. of extract experienced mildly decreased body weights in comparison with the control in non-significant manner, especially at the fourth week. After 28 days of treatment, apart aspartate aminotransferase (AST), no significant changes were found in the blood serum biochemical parameters such as total proteins, alanine aminotransferase (ALT), glucose, creatinin, and lipid levels of the treated mice in comparision with the control group. In view of the current results, A. senegalensis root wood aqueous extract would be considered as safe in both acute and subacute exposure. However, long-term toxicity studies are needed for further toxicological profile elicitation of the plant, and a possible reinforcement of clinical relevance of the results of laboratory studies.