Peganum harmala L. is a medicinal plant growing in the region of Sidi Fredj of Souk Ahras city (Northeastern of Algeria), whose native populations commonly used this plant to treat various health conditions and illnesses. Up to now, no previous study on the beneficial effects of P.harmala in the Souk Ahras region has been conducted. Therefore, the present study aimed to investigate the phenolic and flavonoid composition, as well as the antioxidant activities of the methanol extract and its three fractions: chloroform, ethyl acetate and butanol from seeds of P. harmala using eight different methods. The inhibitory effect of the extracts against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was also determined. The results showed that the ethyl acetate fraction contained the highest amounts of total phenolic and total flavonoid (237.23±1.06 µg GAE/mg and 82.58±0.03 µg QE/mg, respectively). Important antioxidant activity was exerted by the extracts, especially the ethyl acetate fraction, which was the most effective. However, a weak antioxidant activity in Ferrous ions chelation was observed for all extracts with >800 µg/mL. The samples showed also a strong inhibitory effect against AChE and BChE, the chloroform fraction was the most potent with IC50 values of 14.69±0.10 μg/mL against AChE and 7.36±0.77 μg/mL against BChE comparable to that of galantamine. Conclusively, the obtained results proved the beneficial antioxidant and anticholinesterase activities of P. harmala-derived bioactive molecules.