Athenaea velutina (Sendtn.) D’Arcy, a native plant of the Brazilian Atlantic Forest, has exhibited potential pharmacological effects due to the presence of withanolides with anti-cancer and anti-inflammatory activities. However, there is a lack of research on the safety and toxicity of this plant species. This study aimed to assess the safety of the ethanolic extract of A. velutina leaves (Av-E) through toxicity and phytochemical analysis. Acute [1000 mg kg−1 body weight (BW)] and subacute (250, 500, and 1000 mg kg−1 BW) toxicity tests were conducted on female Wistar rats, along with an evaluation of the toxic reversal effect. HPLC-HRMS revealed the presence of withanolide steroids in Av-E. Reduced alanine aminotransferase (ALT), bilirubin levels, and increased calcium concentrations were observed after the acute toxicity test. Subacute toxicity demonstrated lowered ALT (250 and 1000 mg kg−1 BW) and aspartate aminotransferase (AST) (250 and 500 mg kg−1 BW) levels and increased sodium concentration (250 mg kg−1 BW). No fatalities or clinical signs of toxicity occurred, and histological and biochemical analysis revealed no apparent liver or kidney toxicity. In conclusion, Av-E exhibited no toxicity in the experiments, encouraging further research to develop new herbal medicines.