Swine based agro-industries throughout the world are in big threat of new PEDV infection due to lack of efficient prophylactic defenses as well as dependable curing agents. Bioactivity-guided fractionation of a methanol soluble extract from radix of Saposhnikovia divaricata led to the isolation of three new (1−3) together with ten known coumarins (4−13). The structures of new isolates (1−3) were established by extensive spectroscopic analysis and their absolute configurations were assigned based on ECD spectra calculation and analysis. Among all isolates, hyuganin C (5), revealed strongest inhibitory effect on PEDV replication. Quantitative real-time PCR data showed inhibitory effect of 5 on genes responsible for synthesis of PEDV vital structural proteins (GP6 nucleocapsid, GP2 spike, and GP5 membrane) in a dose-dependent manner. Also, compound 5 demonstrated the inhibitory effect on PEDV GP6 nucleocapsid and GP2 spike protein synthesis as analyzed by western blotting. This study represents a new class of chemical entities for developing anti-PEDV agents.