Pajanelia longifolia, a deciduous tree of historical significance, has gained attention for its potential therapeutic properties. This study focuses on evaluating the safety and potential applications of a methanol extract derived from P. longifolia leaves. The investigation covers various critical elements, encompassing GC-MS analysis, evaluation of antioxidant properties, in vitro toxicity assessment, and testing for acute toxicity. The cytotoxicity of plant-derived compounds was verified utilizing the NIH/3T3 cell line through the MTT assay. In the examination of acute toxicity in female Wistar rats, the test subjects were segregated into two categories: a control group and another group that received a single dose of a methanol extract at 2000 mg/kg body weight (b.w.), followed by a monitoring period of 14 days. Analysis using GC-MS identified eighteen distinct chemicals in the methanol extract, while the plant demonstrated notable antioxidant capabilities. The cytotoxicity study revealed minimal toxicity, with an inhibitory concentration (IC50) of 214.95g/mL for the methanol extract. Significantly, the acute toxicity assessment indicated a lethal dose (LD50) exceeding 2000mg/kg body weight, with no observed mortality. Moreover, no noteworthy changes were noted in terms of behaviour, body weight, relative organ weight, haematological parameters, biochemical markers, or histological examinations. These findings suggest that oral administration of the methanol extract from P. longifolia to rats poses minimal risk. Furthermore, this extract holds promise for future therapeutic research endeavours.