In the present scenario, products derived from plants serve as a potential source of anticancer drugs with low toxicity and cost. Lawsonia inermis (LI) has been selected to investigate its antitumor activity due to the increased life span reported by alcoholic root extract in DLA bearing mice. The root of Lawsonia inermis was extracted with 70% alcohol and different fractions viz. LIEA, LIBUT, LIALC and LIAQ were prepared. The LIEA was discovered to have powerful antioxidant properties followed by LIBUT, LIALC and LIAQ among these distinct fractions of root extract prepared. Preliminary screening of cytotoxicity using SRB assay on different cell lines (HCT-116 and HeLa) showed LIEA and LIBUT more potent at 48 h of incubation. Detailed cytotoxicity study on LIEA and LIBUT fractions using various concentrations at different time points demonstrated the more potent activity of LIEA than LIBUT on all the cell lines at 24, 48 and 72 hours. Among the 3 cell lines, HCT-116 was found more sensitive to LIEA with an IC50 of 179 ± 15.5 µg/ml. In vivo study on the EAC mice model showed increased life span in LIEA treated mice at both selected doses (200 mg/kg and 300 mg/kg) and decreased percentage change in the body when compared to EAC bearing mice. LIEA at both the dose reversed the EAC induced alternation in the hematological parameters near to normal. However, 300 mg/kg was found more effective than 200 mg/kg. Thus, this study is an initial step in identification of a novel and selective herbal antitumor agent.