Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread as a pandemic and caused damage to people’s lives and countries’ economies. The spike (S) protein of SARS-CoV-2 contains a cleavage motif, Arg-X-X-Arg, for furin and furin-like enzymes at the boundary of the S1/S2 subunits. Given that cleavage plays a crucial role in S protein activation and viral entry, the cleavage motif was selected as the target. Our previous fluorogenic substrate study showed that osthole, a coumarin compound, inhibits furin-like enzyme activity. In this study, we examined the potential activities of 15 compounds with a structure-activity relationship with osthole, and evaluated their protective ability against SARS-CoV-2 infection. Of the 15 compounds tested, compounds C1 and C2 exhibited the inhibitory effects of osthole against furin-like enzymatic activity; however, little or no inhibitory effects against furin activity were observed. We further examined the inhibition of SARS-CoV-2 activity by compounds C1 and C2 using a Vero E6 cell line that expresses the transmembrane protease serine 2 (TMPRSS2). Compounds C1, C2, and osthole effectively inhibited SARS-CoV-2 infection. Therefore, osthole and its derivatives can potentially be used as therapeutic agents against SARS-CoV-2.