Background: Aegle marmelos belongs to the family Rutaceae, a medium-sized perennial tree which grows in subtropical and tropical parts of India and South-East Asia. Studies have reported that leaf extracts of A. marmelos have anticancer, cardiotonic, antidyslipidemic, and hypoglycemic effects and were ethnologically used for the treatment of dropsy, opthalmitis, ulcers, beri beri, and cholera. Objective: In the present study, we have carried out nontargeted analysis of the leaf extract of A. marmelos Correa ex Roxb., by gas chromatography-mass spectrometry (GC/MS) and estimated the inhibitory concentration (IC50) in human cancer cell lines using MCF-7, H-460, and SF-268. Materials and Methods: Dried leaves of A. marmelos were extracted with 50% aqueous alcohol using Soxhlet apparatus. The dried extract was suspended in water and re-extracted with petroleum ether and the fraction was named as AMPEF (petroleum ether fraction of Aegle marmelos leaf extract); the dry yield of the AMPEF was found to be 7.56% (w/w). Nontargeted GC/MS analysis of AMPEF was performed using Shimadzu QP 2000 GC equipped with ULBON-HR-5 capillary column and mass spectrometer as detector. The cancer cell lines were obtained from the National Centre for Cell Science, Pune. In vitro cytotoxic activity of AMPEF on cancer cell lines was conducted using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. Results: GC/MS analysis of the AMPEF enabled the identification of 26 compounds, and IC50of AMPEF in MCF-7, H-460, and SF-268 was found to be 0.53 ± 0.10, 0.65 ± 0.05, and 0.18 ± 0.01 μg/ml, respectively. Conclusion: The AMPEF was found to be a potential cytotoxic agent against the used cell lines.