In this study, we screened 17 medicinal plants for binding activity to G-quadruplex d(TTGGGTT)(4) by H-1 NMR spectroscopy and found that the crude extract of Peganum harmala L. seeds showed the most potential binding activity. Subsequently, H-1 NMR- and bioassay -guided isolation of the extract of P. harmala L. was performed to obtain four pairs of partially racemized beta-carboline alkaloids, pegaharmines A-D (1-4). Their structures and absolute configurations were determined by extensive NMR analyses, X-ray crystallography, ECD calculations, and CD exciton chirality approaches. Interestingly, pegaharmine D (4), which showed the strongest G-quadruplex interaction, exhibited significant cytotoxic activity against three cancer cell lines. This work contributed a practical strategy for the discovery of novel G-quadruplex ligands from natural products and provided potential insights for using beta-carboline alkaloids as anticancer lead compounds specifically targeting G-quadruplexes.