Currently, SARS-CoV-2 virus is an emerging pathogen that has posed a serious threat to public health worldwide . However, no agents have been approved to treat SARS-CoV-2 infections to date, underscoring the great need for effective and practical therapies for SARS-CoV-2 outbreaks . We reported that a focused screen of OA saponins identified 3-O-β-chacotriosyl OA benzyl ester 2 as a novel small molecule inhibitor of SARS-CoV-2 virus entry, via binding to SARS-CoV-2 glycoprotein (S). We performed structure-activity relationship profiling of 2 and discovered C-17-COOH of OA was an important modification site that improved both inhibitor potency toward SARS-CoV-2 and selectivity index . Then optimization from hit to lead resulted in a potent fusion inhibitor 12f displaying strong inhibition against infectious SARS-CoV-2 with an IC50 value of 0.97 μM in vitro . Mechanism studies confirmed that inhibition of SARS-CoV-2 viral entry of 12f was mediated by the direct interaction with SARS-CoV-2 S2 subunit to block membrane fusion . These 3-O-β-chacotriosyl OA amide saponins are suitable for further optimization as SARS-CoV-2 entry inhibitors with the potential to be developed as therapeutic agents for the treatment of SARS-CoV-2 virus infections.