Nymphaea x rubra Roxb. ex Andrews (N. rubra) has been widely reported for immunomodulatory properties and treatment of piles, bleeding nose and dysentery in traditional medicinal systems. However, its in-vitro and in-vivo toxicity studies have never been investigated. So, the present study was designed to investigate in-vitro and in-vivo toxicity of methanolic extract of N. rubra rhizome in rats. In-vitro cytotoxicity studies were conducted for different doses of extract in N2a cell lines. For in-vivo toxicity studies, SD rats were divided into three groups and administered with 0, 300 and 2000 mg/kg b. w., p. o., of N. rubra extract respectively. In acute toxicity studies, female animals after extract administration animals were sacrificed for hematological profiling and gross necropsy. In sub-acute toxicity studies, both male and female animals were administered with extract daily for 14 days and were sacrificed for hematological, biochemical and histological examination. Body weight and food water intake was measured daily and animals were observed for visual toxic effects, behavioral changes and mortality. During in-vivo toxicity studies, none of the animals showed signs of toxicity and mortality during toxicity studies. The present findings suggest its safety and NOAEL of N. rubra rhizome extract to be > 2000 mg/kg b. w.