This study reports the isolation of four new fl-carboline alkaloids (1-4) and six previously identified alkaloids (5-10) from the roots of Peganum harmala L. Among these compounds, 1 and 2 were characterized as rare fl-carboline-quinazoline dimers exhibiting axial chirality. Compound 3 possessed a unique 6/5/6/7 tetracyclic ring system with an azepine ring, and compound 4 was a novel annomontine fl-carboline. The structures of these compounds were elucidated by spectroscopic data and quantum mechanical calculations. The biosynthetic pathways of 1-3 were proposed. Additionally, the cytotoxicity of some isolates against four cancer cell lines (HL-60, A549, MDA-MB-231, and DU145) was evaluated. Notably, compound 4 exhibited significant cytotoxicity against HL60, A549, and DU145 cells with IC50 values of 12.39, 12.80, and 30.65 mu mol center dot L-1, respectively. Furthermore, compound 2 demonstrated selective cytotoxicity against HL-60 cells with an IC50 value of 17.32 mu mol center dot L-1.