CAPES, CNPq, FAPEAM and FINEP for the financial support, and Gabriel G. Gato for helping in organizing the IR tables.A combined experimental and theoretical DFT study of the structural, vibrational and electronic properties of 9-methoxyguatterfriesine (I), (R)-6,6a-dihydro-9-methoxyguatterfriesine (II) and 4,5-dehydro- 9-methoxyguatterfriesine (III) is presented using B3LYP exchange-correlation functional with 6-311G (2d, p) basis set. The theoretical geometry optimization data were compared with the X-ray data for ( )-N-acetylanonaine, showing close values. In addition, molecular electrostatic potential surface (MEPS) calculation, HOMO-LUMO energy gap, natural bond orbitals (NBOs) and first and second order hyperpolarizabilities were also performed with the same calculation level. Transitions for UV spectrum for the three structures were assigned and the calculated bands showed good agreement with the measured experimental data. The comparative IR studies showed intermolecular hydrogen bonds that stabilize dimeric forms proposed for the three molecules and also revealed several characteristic vibrations for the structures. Molecular docking studies with DNA Topoisomerase II-DNA complex showed binding free energies of 7.6, 7.5 and 8.7 kcal/mol, for I, II and III respectively, which indicate III as a better potential inhibitor for this enzyme. In vitro cytotoxicity assay revealed an expressive antitumor activity of III against HepG2 cell line.