COVID-19 caused by SARS-CoV-2 has posed a significant threat to global public health since its outbreak in late 2019. Although there are a few drugs approved for clinical treatment to combat SARS-CoV-2 infection currently, the severity of the ongoing global pandemic still urges the efforts to discover new antiviral compounds. As the viral spike (S) protein plays a key role in mediating virus entry, it becomes a potential target for the design of antiviral drugs against COVID-19. Here, we tested the antiviral activity of berbamine hydrochloride, a bis-benzylisoquinoline alkaloid, against SARS-CoV-2 infection. We found that berbamine hydrochloride could efficiently inhibit SARS-CoV-2 infection in different cell lines. Further experiments showed berbamine hydrochloride inhibits SARS-CoV-2 infection by targeting the viral entry into host cells. Moreover, berbamine hydrochloride and other bis-benzylisoquinoline alkaloids could potently inhibit S-mediated cell-cell fusion. Furthermore, molecular docking results implied that the berbamine hydrochloride could bind to the post fusion core of SARS-CoV-2 S2 subunit. Therefore, berbamine hydrochloride may represent a potential efficient antiviral agent against SARS-CoV-2 infection.