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Highlights • Sambucus FormosanaNakai extract reduced cytopathicity and virus yield in HCoV-NL63-infected cells. • Among phenolic acid constituents, caffeic acid, chlorogenic acid and gallic acid sustained the anti-HCoV-NL63 activity. • Sambucus FormosanaNakai extract and caffeic acid concentration-dependently inhibited HCoV-NL63 attachment onto cells., Human coronavirus NL63 (HCoV-NL63), one of the main circulating HCoVs worldwide, causes respiratory tract illnesses like runny nose, cough, bronchiolitis and pneumonia. Recently, a severe respiratory illness outbreak of HCoV-NL63 has been reported in a long-term care facility. Sambucus FormosanaNakai, a species of elderberry, is a traditional medicinal herb with anti-inflammatory and antiviral potential. The study investigated the antiviral activity of Sambucus FormosanaNakai stem ethanol extract and some phenolic acid constituents against HCoV-NL63. The extract was less cytotoxic and concentration-dependently increased anti-HCoV-NL63 activities, including cytopathicity, sub-G1 fraction, virus yield (IC50 = 1.17 μg/ml), plaque formation (IC50 = 4.67 μg/ml) and virus attachment (IC50 = 15.75 μg/ml). Among the phenolic acid constituents in Sambucus FormosanaNakai extract, caffeic acid, chlorogenic acid and gallic acid sustained the anti-HCoV-NL63 activity that was ranked in the following order of virus yield reduction: caffeic acid (IC50 = 3.54 μM) > chlorogenic acid (IC50 = 43.45 μM) > coumaric acid (IC50 = 71.48 μM). Caffeic acid significantly inhibited the replication of HCoV-NL63 in a cell-type independent manner, and specifically blocked virus attachment (IC50 = 8.1 μM). Therefore, the results revealed that Sambucus Formosana Nakai stem ethanol extract displayed the strong anti-HCoV-NL63 potential; caffeic acid could be the vital component with anti-HCoV-NL63 activity. The finding could be helpful for developing antivirals against HCoV-NL63.