Ethnopharmacological relevance: Piper cemuum Veil (Piperaceae) is a native species from Atlantic rain forest, popularly known as pariparoba. Its leaves have been commonly used by rural and urban communities from State of Sao Paulo, Brazil, to treat pain (orally and topically), and hepatic and renal complications. Aim of the study: In this study we evaluated the acute and sub-acute toxicity, genotoxicity and mutagenicity of hydroalcoholic extract obtained from P. cemuum leaf using in vivo and in vitro methods. Material and methods: In the acute toxicity study, mice were orally treated with P. cemuum extract (2000 mg/kg, p.o.). General behavior and mortality were observed for up to 14 days. In the sub-acute toxicity study, P. cemuum extract was given orally as a single administration to the rats at doses of 50 or 250 mg/kg/day, for 28 days. General behavior, body weight, biochemical and hematological parameters, organ coefficients and pathological morphology were analyzed. The P. cemuum mutagenicity was evaluated using mammalian cell micronucleus assay. Additionally, in vitro toxicity profile of the extract was assessed through cytotoxicity, hemolytic activity, and genotoxicity assay. Results: Data from comet assay demonstrates that high concentrations of P. cemuum extract induce genotoxicity. However, no evidence of hemolytic, cytotoxic or mutagenicity activity was found. In addition, the acute and subacute toxicity studies did not show significant changes in body weight, general behavior, hematology and biochemical parameters, organ weight and liver and kidney histopathological analysis. Conclusions: Together, the results herein obtained indicate that P. cemuum leaves extract did not present significant toxicity when administered to male or female rats. Additionally, no significant alteration in hematological, biochemical and morphological parameters were observed. Data obtained in vitro shows that extract did not present cytotoxicity and mutagenicity. However, the extract induces in vitro genotoxicity, but in high concentration. Further studies are necessary to evaluate the safety of long-term exposure to P. cemuum leaves extract added to in vivo genotoxicity.