Isoquinoline alkaloidal content of Berberis vulgaris L. (Berberidaceae) is regarded as the main source of pharmacological activity. Studies showed that berberine (BB) possesses cytotoxic and hepatoprotective properties. The aim of the study was to isolate BB from B. vulgaris and semisynthesis berberrubine (M1) and diberberine sulphate (Di-BB), investigate their efficiency as cytotoxic, antihepatitis C virus (HCV) agents and acetylcholinesterase enzyme (AChE) inhibitors. M1 and Di-BB were semisynthesized from BB previously isolated from Berberis vulgaris methanolic extract. Structures of the three compounds were elucidated on basis of 1H- and 13C-NMR and LC/MS. Cytotoxicity activity was studied using both normal peripheral blood mononuclear cells as control and three different proliferating cancerous cell lines; HepG-2, Caco-2 and MCF-7. The capability to inhibit viral replication was evidenced by the disappearance of viral RNA-amplified products detected by RT-PCR after incubation for 96 h. In vitro study of cytotoxic activities showed that, B. vulgaris extract was the most efficient on Caco-2 (IC50 3.836 μg/mL) and HepG-2 (IC50 5.55 μg/mL) cells lines, while, BB was the most potent on MCF-7 (IC50 4.433μg/mL) at 48 h. Replication of HCV RNA was inhibited at 100 μg/mL of each of B. vulgaris extract, BB and Di-BB. Finally, Di-BB significantly inhibited AChE activity (IC50: 100 μg/mL). In conclusion, the alkaloids studied possessed potent hepato-protective properties. Furthermore, the AChE inhibiting activity of Di-BB makes it a point of interest for further study on treatment of Alzheimer's disease.